Radioligands for pretargeted pet imaging and methods of their therapeutic use

ABSTRACT

Described herein are Tz/TCO-based pretargeting strategies using an Al[ 18 F]-NOTA-labeled tetrazine radioligand. This imaging strategy enables delineation of cancer at earlier time points compared to other imaging strategies and further decreases the radiation dose to healthy tissues compared to directly labeled antibodies. Al-based  18 F imaging of small molecules, such as tetrazine, has not been previously achieved due to the decomposition of tetrazine during radiofluorination. Radiofluorination is advantageous over other radiolabeling methods because, in addition to having a shorter half-life,  18 F is more readily available to produce and therefore integrated into hospital workflows.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of, and incorporatesherein by reference in its entirety, U.S. Provisional Patent ApplicationNo. 62/159,763, filed May 11, 2015.

GOVERNMENT SUPPORT

This invention was made with government support under grant 1K99CA178205-01 awarded by the National Institutes of Health (NIH)/NationalCancer Institute (NCI) and grants P30 CA08748, K25 EB016673, TR; R00CA1440138, BMZ; 2R42CA128362, awarded by the National Institutes ofHealth (NIH). The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to pretargeted positron emissiontomography (PET) imaging of cancer. In particular embodiments, theinvention relates to radioligands for pretargeted PET imaging of cancer(e.g., colorectal, pancreatic, etc.).

BACKGROUND

Over the past 25 years, antibodies have emerged as effective vectors forthe sensitive and specific delivery of radioisotopes to tumors. A widevariety of radionuclides ranging from 1241 for positron emissiontomography (PET) to 225Ac for targeted radiotherapy have been conjugatedto antibodies for preclinical investigations and clinical applications.However, the size and dosing of these radiolabeled antibodies haveobstructed clinical utility. For example, intact IgG antibodies haverelatively long biological half-lives due to their size, which causesthem to require multiple days to weeks to reach their optimalbiodistribution. Consequently, the large size of intact IgGs requiresisotopes with long physical half-lives, such as ⁸⁹Zr (t_(1/2)=3.26 d) or²⁴I (t_(1/2)=4.2 d) for PET or ¹⁷⁷Lu (t_(1/2)=6.73 d) for radiotherapy.While the resulting radioimmunoconjugates can deliver highconcentrations of activity to tumor tissue, the long circulation timesand radioactive half-lives combine to produce significant andpotentially deleterious radiation doses to healthy tissues.

To overcome these limitations, radiopharmaceuticals based on truncatedimmunoglobulins, such as F(ab′)2, F(ab′), and scFv fragments have beendeveloped to provide imaging agents that combine the specificity andaffinity of intact IgGs with the rapid pharmacokinetics and favorabledosimetry of smaller molecules. However, preclinical studies have shownthat the smaller size and more rapid pharmacokinetics result indecreased activity concentrations in the targeted tissue in addition toincreased activity concentrations in excretory organs such as thekidneys.

To address these limitations, pretargeting, or strategies to decouplethe antibody from the radioactivity at the time of injection, have beendeveloped to provide an alternative approach to harness the highaffinity and specificity of intact IgGs, while avoiding theirpharmacokinetic drawbacks. Pretargeting uses an antibody designed tobind both a target antigen and a radiolabeled hapten. The antibody isinjected into the blood and, in turn, is given time to accumulate at thetumor and concomitantly clear from the body. After the accumulation, aradiolabeled hapten is administered intravenously. The radioligandeither binds to the antibody at the tumor or, due to its small size,rapidly clears from the body. If the radioligand binds to the antibody,the final radioimmunconjugate is formed at the tumor site. Thus,pretargeting achieves delineation of tumor tissue at much earlier timepoints than traditional radioimmunoconjugates while significantlyreducing the overall radiation burden to the patient.

Until recently, three types of pretargeting technologies have been used:(1) streptavidin-fused antibodies and biotin-based radioligands, (2)oligonucleotide-labeled antibodies and radioligands bearingcomplementary sequences, and (3) bispecific antibodies that bind both atarget antigen and a radiolabeled hapten (e.g., ⁹⁰Y-DOTA). Yet despitesignificant preclinical successes, the widespread application of eachstrategy has been hampered by its intrinsic limitations. For example,the immunogenicity of the streptavidin-bearing immunoconjugates or theinherent lack of modularity of bispecific antibodies limits use inclinics.

Over the last few years, a pretargeting strategy based on the inverseelectron demand Diels-Alder (IEDDA) reaction between tetrazine (Tz) andtranscyclooctene (TCO) was developed and is depicted in FIG. 1. TheIEDDA cycloaddition as a click chemistry conjugation reaction has beenused for a variety of applications, including the fluorescent labelingof nanoparticles, antibodies, oligonucleotides, and small molecules, aswell as the traditional synthesis of radiopharmaceuticals.

The IEDDA reaction was thought to be a promising tool for in vivopretargeting. For example, the IEDDA reaction is extraordinarily rapid(k₁ greater than 30,000 M⁻¹ s⁻¹), selective, robust, and bioorthogonal.The use of IEDDA in pretargeting was pioneered largely by Rossin, etal., who published an ¹¹¹In-based SPECT imaging approach and haveimproved their systems to use tetrazine-bearing clearing agents and morereactive dienophiles (Rossin, R.; van den Bosch, S. M.; Ten Hoeve, W.;Carvelli, M.; Versteegen, R. M.; Lub, J.; Robillard, M. S., Highlyreactive trans-cyclooctene tags with improved stability for Diels-Alderchemistry in living systems. Bioconj. Chem. 2014, 34, 1210-1217).

An alternative pretargeting PET imaging strategy based on the IEDDAreaction was reported by Zeglis, B. M. et al. Journal of NuclearMedicine. 54, 1389-1396 (2013) and in U.S. Provisional Application No.62/159,763, which are hereby incorporated by reference herein in theirentireties. This strategy featured a rapid and selective in vivobiorthogonal reaction between trans-cyclooctene (TCO) and tetrazine(Tz). The use of antibodies labeled with TCO and small moleculetetrazine-based radioligands allowed radioactive labeling of antibodiesafter accumulation at the target site in vivo. However, one of thedrawbacks of this system was that the ⁶⁴Cu-NOTA-labeled tetrazineradioligand cleared too slowly from the gut (e.g., small intestine,large intestine, etc.). Although the pretargeting system delineatedcancer tumors with this radioligand, later imaging time points (e.g., at12 hours (h)) were required to achieve high image contrast, and thedosimetric benefits of the system were not significant. Moreover,unclicked ⁶⁴Cu-Tz-NOTA radioligand was cleared sluggishly through theintestines, hindering use as a clinical imaging system for cancer (e.g.,colorectal) detection. The U.S. Provisional Application No. 62/159,763reports a ⁶⁴Cu-labeled radioligand employed for the pretargeted PETimaging of SW1222 human colorectal cancer xenografts.

As the half-life of ⁶⁴Cu is 12.74 hours the potential of utilizing evenshorter-lived radionuclides to decrease the time period between theadministration of the radiotracer and imaging (e.g., generate an imageat an earlier time point) has significant clinical benefits: The abilityto acquire clinical imaging data at earlier time points reducesradiation exposure to the patient and expedites the time that thepatient is in the hospital.

As mentioned above, various pretargeting strategies based on the IEDDAreaction between Tz and TCO have been developed using ⁶⁴Cu-basedapproaches. In addition to the aforementioned advantages of short-livedradionuclides, the availability of ¹⁸F (or ⁶⁸Ga) is considerably highercompared to ⁶⁴Cu for instance as most hospitals and imaging centers donot produce ⁶⁴Cu locally and rely on the delivery from external sources.In contrast to ⁶⁴Cu, most hospitals and imaging centers have the abilityto produce the ¹⁸F radionuclide in house and hence make the use of¹⁸F-labeled radiotracer logistically more desirable.

Although ¹⁸F-incorporation into the Tz moiety has been attempted (Li, Z,et al., Chem. Commun., 2010, 46, 8043-8045, Li, Z, et al., Chem.Commun., 2010, 46, 8043-8045, Reiner and Zeglis, J. Label Compd.Radiopharm, 2013), this has not been successful, and workarounds havebeen necessary, for example, attaching the ¹⁸F radionuclide to TCO ordextran. The radiosynthesis of ¹⁸F attached to a small molecule, such asan ¹⁸F-labeled tetrazine moiety capable of pretargeted in vivo imaging,has not been published.

Therefore, there is a need to develop low-dose radioimmunoconjugatesthat delineate cancer from healthy tissue in small amounts of time,possess favorable pharmacokinetic profiles, can be cleared rapidly fromthe body to enable high contrast PET imaging of cancer (e.g.,colorectal, pancreatic), and are commercially available to orsynthesized by hospitals.

SUMMARY OF INVENTION

Described herein are Tz/TCO-based pretargeting strategies using (1) a⁶⁴Cu-sarcophagine-based tetrazine radioligand for pretargeted PETimaging with more rapid excretion of the excess radioligand through thebladder and kidneys and (2) an Al[¹⁸F]-NOTA-labeled tetrazineradioligand. These imaging strategies enable delineation of cancer atearlier time points compared to other imaging strategies and furtherdecrease the radiation dose to healthy tissues compared to directlylabeled antibodies.

The present disclosure describes the development and in vivo validationof an improved strategy for pretargeted PET imaging of colorectal andpancreatic cancer.

In certain embodiments, the present disclosure describes the synthesisand characterization of in vivo behavior of two ⁶⁴Cu-labeled tetrazineradioligands: ⁶⁴Cu-Tz-PEG₇-NOTA and ⁶⁴Cu-Tz-SarAr. These tworadioligands possess structural alterations that modulate theirpharmacokinetic profile compared to prior imaging systems. In someexample embodiments, the system described herein produces higheractivity concentrations in the tumor and, due to the renal excretion ofthe unbound radioligand disclosed herein, provides improvedtumor-to-background activity concentration ratios at early time points.Furthermore, the presently disclosed methodologies function atsignificantly lower dose rates required by directly labeledradioimmunoconjugates. In some certain embodiments, the present imagingstrategies can be effective for any cancer (e.g., colorectal,pancreatic) for which a non-internalizing, biomarker-targeted antibodyexists.

In certain embodiments, a TCO-bearing immuno-conjugate of theanti-CA19.9 antibody 5B1 and an Al[¹⁸F]-NOTA-labeled tetrazineradioligand were harnessed for the visualization of CA19.9-expressingBxPC3 pancreatic cancer xenografts. Al-based ¹⁸F imaging of smallmolecules, such as tetrazine, has not been previously achieved due tothe decomposition of tetrazine during radiofluorination.Radiofluorination is advantageous over other radiolabeling methodsbecause, in addition to having a shorter half-life, ¹⁸F is more readilyavailable to produce, and therefore, more suited to be integrated intohospital workflows. Biodistribution and ¹⁸F-PET imaging data clearlydemonstrate that this methodology effectively delineates tumor mass withactivity concentrations up to 6.4% ID/g at 4 h after injection of theradioligand.

In one aspect, the invention is directed to a composition comprising: atetrazine moiety (Tz); a radiolabel (e.g., 18F); a chelator (e.g.,1,4,7-triazonane-1,4,7-triyl-triacetic acid (NOTA), e.g.,1,4,7-triazacyclononane-1,4-diacetate (NODA)); a linker (e.g.,polyethylene glycol, (poly)-L-lysine) attaching the tetrazine moiety(Tz) to the chelator; and aluminum or aluminum-containing moiety (e.g.,wherein the radiolabel is attached to aluminum).

In certain embodiments, the linker is polyethylene glycol (PEG) or(poly)-L-lysine and has a length of from 1 to 100 units and 1 to 200units, respectively.

In certain embodiments, the composition comprises:

In another aspect, the invention is directed to a radioimmunoconjugatecomprising: (1) a targeting moiety-transcyclooctene (TCO) conjugate(e.g., TCO-5B1); and (2) a radioligand (e.g., attached to the targetingmoiety-TCO conjugate) comprising a tetrazine moiety (Tz); a radiolabel(e.g., 18F); a chelator (e.g., 1,4,7-triazonane-1,4,7-triyl-triaceticacid (NOTA), e.g., 1,4,7-triazacyclononane-1,4-diacetate (NODA)); alinker (e.g., polyethylene glycol, (poly)-L-lysine) attaching thetetrazine moiety (Tz) to the chelator; and aluminum oraluminum-containing moiety (e.g., wherein the radiolabel is attached toaluminum).

In certain embodiments, the tetrazine moiety (Tz), the chelator, and thelinker attaching the tetrazine moiety to the chelator comprises a memberselected from the group consisting of:2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-1-oxo-5,8,11,14,17,20,23-heptaoxa-2-azapentacosan-25-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35-undecaoxa-2-azaheptatriacontan-37-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′-(7-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35-undecaoxa-2-azaheptatriacontan-37-yl)thioureido)benzyl)-1,4,7-triazonane-1,4-diyl)diaceticacid;2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,7-dioxo-11,14,17,20,23,26,29-heptaoxa-2,8-diazahentriacontan-31-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,7-dioxo-11,14,17,20,23,26,29,32,35,38,41-undecaoxa-2,8-diazatritetracontan-43-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(25,28-dioxo-28-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)-3,6,9,12,15,18,21-heptaoxa-24-azaoctacosyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(37,40-dioxo-40-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36-azatetracontyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)-3-oxo-6,9,12,15,18,21,24-heptaoxa-2-azaheptacosan-27-amido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(2-(4-(1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy)-3,6,9,12,15,18,21,24,27,30,33-undecaoxahexatriacontan-36-amido)benzyl)-1,4,7-triazonane-1,4,7-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(5-amino-6-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′-(7-(4-(3-(5-amino-6-((4-6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-triazonane-1,4-diyl)diaceticacid;2,2′,2″-(3-(4-(3-(5-amino-6-((5-amino-6-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid; and2,2′,2″-(3-(4-(3-(5-amino-6-((5-amino-6-((5-amino-6-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)amino)-6-oxohexyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid.

In certain embodiments, the composition is hydrophilic (e.g., having apartition coefficient less than 2).

In certain embodiments, the radioligand comprises:

In certain embodiments, the targeting moiety-TCO conjugate has a TCOmoiety comprises:

In certain embodiments, wherein linker is polyethylene glycol (PEG) or(poly)-L-lysine and has a length of from 1 to 100 units and 1 to 200units, respectively.

In another aspect, the invention is directed to a method forsynthesizing a radioligand: (1) preparing Tz-PEG11-NOTA(tetrazine-polyethylene glycol-1,4,7-triazonane-1,4,7-triyl-triaceticacid) (e.g., comprising coupling Tz-NHS (tetrazine-N-hydroxysuccinimide)to NH2-PEG11-NHBoc; deprotecting the terminal tert-butyloxycarbonylprotecting group to obtain Tz-PEG11-NH2; and reacting Tz-PEG11-NH2 withp-SCN-Bn-NOTA to yield Tz-PEG11-NOTA (e.g., wherein Tz-PEG11-NOTA isprepared with a purity of greater than 98% and with an overall yield ofgreater than 10%)); (2) preparing a Al-¹⁸F complex (e.g., comprisingeluting [18F]fluoride into a metal-free solvent; mixing the eluted[18F]fluoride with a AlCl3-solution (e.g., wherein mixing is performedat room temperature and a pH of 3.5-4); incubating the mixture (e.g.,wherein the mixture is incubated at 30° C.); and (3) reacting theTz-PEG11-NOTA (e.g., wherein the solvent of the prepared Tz-PEG11-NOTAis acetonitrile (MeCN)/water in a volumetric ratio of 3:1) and theAl-¹⁸F complex to yield the radioligand Tz-PEG11-Al[¹⁸F]-NOTA, whereinthe radioligand is obtained in 54% to 65% radiochemical yield(decay-corrected to the start of synthesis) with a purity greater than96% and specific activities between 20 to 30 Gbq/μmol.

In another aspect, the invention is directed to a method for detectingtumor cells, the method comprising: (1) administering (e.g., injecting)a quantity of targeting moiety-transcyclooctene (TCO) conjugate (e.g.,TCO-5B1) to a subject, wherein a portion of the targeting moiety-TCOconjugate localizes at the tumor cells and unbound targeting moiety-TCOconjugate is cleared (e.g., from the blood, from the renal system,and/or from the subject) after an accumulation interval (e.g., whereinthe accumulation interval is less than 240 hours, e.g., less than 216hours, e.g., less than 192 hours, e.g., less than 144 hours, e.g., lessthan 120 hours, e.g., less than 96 hours, e.g., less than 72 hours,e.g., less than 48 hours, e.g., less than 24 hours); (2) administering(e.g., injecting) a radioligand to the subject after the accumulationinterval, wherein the radioligand comprises a tetrazine moiety (Tz); aradiolabel (e.g., 18F); a chelator (e.g.,1,4,7-triazonane-1,4,7-triyl-triacetic acid (NOTA), e.g.,1,4,7-triazacyclononane-1,4-diacetate (NODA)); a linker (e.g.,polyethylene glycol, (poly)-L-lysine) attaching the tetrazine moiety(Tz) to the chelator; and aluminum or aluminum-containing moiety (e.g.,wherein the radiolabel is attached to aluminum), wherein the targetingmoiety-TCO conjugate and the radioligand bind together to form aradioimmunoconjugate via an in vivo click reaction at the tumor sitewithin a region of the subject; and (3) imaging (e.g., via positronemission tomography (PET) imaging) the radioimmunoconjugate accumulatedin the region of the subject within a time period less than 9 hours(e.g., less than 6 hours, less than 4 hours, less than 2 hours) from theadministering of the radioligand.

In certain embodiments, the radioligand comprises:

In certain embodiments, the targeting moiety-TCO conjugate has a TCOmoiety comprising:

In certain embodiments, the radioligand has an effective dose of lessthan 0.1 rem/mCi (e.g., less than 0.05 rem/mCi) over a 4 houraccumulation interval.

In certain embodiments, the radioligand has a half-life in blood that isless than 100 minutes.

In certain embodiments, the radioimmunoconjugate has an activityconcentration in a large intestine of the subject that is less than 2%of the initial dose per gram (ID/g) (e.g., less than 1% ID/g, e.g., lessthan 0.5% ID/g, e.g., less than 0.1% % ID/g) after 2 hour postinjection.

In certain embodiments, the radioimmunoconjugate has an activityconcentration in a gastrointestinal tract of the subject that is lessthan 2% of the initial dose per gram (ID/g) (e.g., less than 1% ID/g,less than 0.5% ID/g, e.g., less than 0.1% % ID/g) after 2 hour postinjection.

In certain embodiments, the radioimmunoconjugate has an activityconcentration in a hepatobiliary system of the subject that is less than2% of the initial dose per gram (ID/g) (e.g., less than 1% ID/g, lessthan 0.5% ID/g, e.g., less than 0.1% % ID/g) after 2 hour postinjection.

In certain embodiments, the linker is polyethylene glycol (PEG) or(poly)-L-lysine and has a length of from 1 to 100 units and 1 to 200units, respectively.

In certain embodiments, the radioligand is hydrophilic (e.g., whereinthe hydrophilicity of the composition is determined by a partitioncoefficient (e.g., wherein the partition coefficient is less than 2)).

In certain embodiments, the targeting moiety is an antibody. In certainembodiments, the antibody is a member selected from the group consistingof trastuzumab, J591, bevacizumab, B43.13, AR9.6, 3F8, 8H9, huA33, and5B1. In certain embodiments, the targeting moiety is a nanoparticle, apeptide, or other biomolecule.

In certain embodiments, the tumor cells are colorectal tumor cells orpancreatic tumor cells (e.g., wherein the tumor cells can be detectedusing a non-internalizing, biomarker-targeted antibody) (e.g., whereinthe tumor cells comprise a biomarker on the surface of the cell).

In certain embodiments, the tetrazine moiety (Tz), the chelator, and thelinker attaching the tetrazine moiety to the chelator comprises a memberselected from the group consisting of:2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-1-oxo-5,8,11,14,17,20,23-heptaoxa-2-azapentacosan-25-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35-undecaoxa-2-azaheptatriacontan-37-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′-(7-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35-undecaoxa-2-azaheptatriacontan-37-yl)thioureido)benzyl)-1,4,7-triazonane-1,4-diyl)diaceticacid;2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,7-dioxo-11,14,17,20,23,26,29-heptaoxa-2,8-diazahentriacontan-31-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,7-dioxo-11,14,17,20,23,26,29,32,35,38,41-undecaoxa-2,8-diazatritetracontan-43-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(25,28-dioxo-28-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)-3,6,9,12,15,18,21-heptaoxa-24-azaoctacosyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(37,40-dioxo-40-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36-azatetracontyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)-3-oxo-6,9,12,15,18,21,24-heptaoxa-2-azaheptacosan-27-amido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(2-(4-(1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy)-3,6,9,12,15,18,21,24,27,30,33-undecaoxahexatriacontan-36-amido)benzyl)-1,4,7-triazonane-1,4,7-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(5-amino-6-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′-(7-(4-(3-(5-amino-6-((4-6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-triazonane-1,4-diyl)diaceticacid;2,2′,2″-(3-(4-(3-(5-amino-6-((5-amino-6-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid; and2,2′,2″-(3-(4-(3-(5-amino-6-((5-amino-6-((5-amino-6-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)amino)-6-oxohexyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid.

In another aspect, the invention is directed to a kit for targetedpositron emission tomography (PET) comprising: a plurality ofcontainers, wherein each container has a type selected from an ampule, avial, a cartridge, a reservoir, a lyo-ject, or a pre-filled syringe; thecomposition; and a targeting moiety-transcyclooctene (TCO) conjugate(e.g., TCO-5B1), wherein a first container of the plurality ofcontainers holds (e.g., contains) the composition (e.g., a firstsolution comprising the composition); and a second container of theplurality of containers holds (e.g., contains) the targeting moiety-TCOconjugate (e.g., a second solution comprising the conjugate).

DEFINITIONS

In order for the present disclosure to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

In this application, the use of “or” means “and/or” unless statedotherwise. As used in this application, the term “comprise” andvariations of the term, such as “comprising” and “comprises,” are notintended to exclude other additives, components, integers or steps. Asused in this application, the terms “about” and “approximately” are usedas equivalents. Any numerals used in this application with or withoutabout/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

“Administration”: The term “administration” refers to introducing asubstance into a subject. In general, any route of administration may beutilized including, for example, parenteral (e.g., intravenous), oral,topical, subcutaneous, peritoneal, intraarterial, inhalation, vaginal,rectal, nasal, introduction into the cerebrospinal fluid, orinstillation into body compartments. In some embodiments, administrationis oral. Additionally or alternatively, in some embodiments,administration is parenteral. In some embodiments, administration isintravenous.

“Biocompatible”: The term “biocompatible”, as used herein is intended todescribe materials that do not elicit a substantial detrimental responsein vivo. In certain embodiments, the materials are “biocompatible” ifthey are not toxic to cells. In certain embodiments, materials are“biocompatible” if their addition to cells in vitro results in less thanor equal to 20% cell death, and/or their administration in vivo does notinduce inflammation or other such adverse effects. In certainembodiments, materials are biodegradable.

“Biodegradable”: As used herein, “biodegradable” materials are thosethat, when introduced into cells, are broken down by cellular machinery(e.g., enzymatic degradation) or by hydrolysis into components thatcells can either reuse or dispose of without significant toxic effectson the cells. In certain embodiments, components generated by breakdownof a biodegradable material do not induce inflammation and/or otheradverse effects in vivo. In some embodiments, biodegradable materialsare enzymatically broken down. Alternatively or additionally, in someembodiments, biodegradable materials are broken down by hydrolysis. Insome embodiments, biodegradable polymeric materials break down intotheir component polymers. In some embodiments, breakdown ofbiodegradable materials (including, for example, biodegradable polymericmaterials) includes hydrolysis of ester bonds. In some embodiments,breakdown of materials (including, for example, biodegradable polymericmaterials) includes cleavage of urethane linkages.

“Biomolecule”: As used herein, “biomolecule” refers to bioactive,diagnostic, and prophylactic molecules. Biomolecules that can be used inthe present invention include, but are not limited to, synthetic,recombinant or isolated peptides and proteins such as antibodies andantigens, receptor ligands, enzymes, and adhesion peptides; nucleotidesand polynucleic acids such as DNA and antisense nucleic acid molecule;activated sugars and polysaccharides; bacteria; viruses; and chemicaldrugs such as antibiotics, antiinflammatories, and antifungal agents.

“Carrier”: As used herein, “carrier” refers to a diluent, adjuvant,excipient, or vehicle with which the compound is administered. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.Water or aqueous solution saline solutions and aqueous dextrose andglycerol solutions are preferably employed as carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

“Radiolabel”: As used herein, “radiolabel” refers to a moiety comprisinga radioactive isotope of at least one element. Exemplary suitableradiolabels include but are not limited to those described herein. Insome embodiments, a radiolabel is one used in positron emissiontomography (PET). In some embodiments, a radiolabel is one used insingle-photon emission computed tomography (SPECT). In some embodiments,radioisotopes comprise ^(99m)Tc, ¹¹¹In, ⁶⁴Cu, ⁶⁷Ga, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm,¹⁷⁷Lu, ⁶⁷Cu, ¹²³I, ¹²⁴I, ¹²⁵I, ¹¹C, ¹3N, ¹⁵O, ¹⁸F, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm,¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹Pm, ⁹⁰Y, ²¹³Bi, ¹⁰³Pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au,¹⁹⁹Au, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, ⁸⁹Zr, ²²⁵Ac, and¹⁹²Ir.

“Subject”: As used herein, the term “subject” includes humans andmammals (e.g., mice, rats, pigs, cats, dogs, and horses). In manyembodiments, subjects are be mammals, particularly primates, especiallyhumans. In some embodiments, subjects are livestock such as cattle,sheep, goats, cows, swine, and the like; poultry such as chickens,ducks, geese, turkeys, and the like; and domesticated animalsparticularly pets such as dogs and cats. In some embodiments (e.g.,particularly in research contexts) subject mammals will be, for example,rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine suchas inbred pigs and the like.

“Small molecule”: As used herein, the term “small molecule” can refer toa non-polymeric molecule, for example, or a species less than 5000 Da.

“Therapeutic agent”: As used herein, the phrase “therapeutic agent”refers to any agent that has a therapeutic effect and/or elicits adesired biological and/or pharmacological effect, when administered to asubject.

“Treatment”: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a substance that partiallyor completely alleviates, ameliorates, relives, inhibits, delays onsetof, reduces severity of, and/or reduces incidence of one or moresymptoms, features, and/or causes of a particular disease, disorder,and/or condition. Such treatment may be of a subject who does notexhibit signs of the relevant disease, disorder and/or condition and/orof a subject who exhibits only early signs of the disease, disorder,and/or condition. Alternatively or additionally, such treatment may beof a subject who exhibits one or more established signs of the relevantdisease, disorder and/or condition. In some embodiments, treatment maybe of a subject who has been diagnosed as suffering from the relevantdisease, disorder, and/or condition. In some embodiments, treatment maybe of a subject known to have one or more susceptibility factors thatare statistically correlated with increased risk of development of therelevant disease, disorder, and/or condition.

Drawings are presented herein for illustration purposes, not forlimitation.

BRIEF DESCRIPTION OF DRAWINGS

The foregoing and other objects, aspects, features, and advantages ofthe present disclosure will become more apparent and better understoodby referring to the following description taken in conduction with theaccompanying drawings, in which:

FIG. 1 shows the inverse electron demand Diels-Alder cycloaddition.

FIG. 2 shows a schematic of the pretargeted PET imaging strategy.

FIG. 3A shows a structure of ⁶⁴Cu-Tz-NOTA.

FIG. 3B shows a structure of ⁶⁴Cu-Tz-PEG₇-NOTA.

FIG. 3C shows a structure of ⁶⁴Cu-Tz-SarAr.

FIG. 4 shows a schematic of the synthesis of Tz-PEG₇-NOTA.

FIG. 5 shows a schematic of the synthesis of Tz-SarAr.

FIG. 6 shows a schematic of the radiosynthesis of ⁶⁴Cu-Tz-PEG₇-NOTA.

FIG. 7 shows a schematic of the radiosynthesis of ⁶⁴Cu-Tz-SarAr.

FIG. 8 shows a crude radio-HPLC chromatogram of ⁶⁴Cu-Tz-PEG₇-NOTA.

FIG. 9 shows a crude radio-HPLC chromatogram of ⁶⁴Cu-Tz-SarAr.

FIG. 10 shows a schematic of the synthesis of huA33-TCO

FIG. 11 shows biodistribution data (% ID/g±SD) of ⁶⁴Cu-Tz-NOTA,⁶⁴Cu-Tz-PEG₇-NOTA, and ⁶⁴Cu-Tz-SarAr in healthy athymic nude (n=4 foreach time point). Mice were administered the radioligands (25-30 μCi in200 mL 0.9% sterile saline) via intravenous tail vein injection andeuthanized by CO₂(g) asphyxiation at 1, 4, and 24 h after injection.

FIG. 12 shows PET imaging data for ⁶⁴Cu-Tz-NOTA in two healthy mice.Healthy female athymic nude mice were administered ⁶⁴Cu-Tz-NOTA (300-350μCi in 200 mL 0.9% sterile saline) via intravenous tail vein injection(t=0). Approximately 5 minutes prior to the PET images, mice wereanesthetized by inhalation of 2% isoflurane (Baxter Healthcare,Deerfield, Ill.)/oxygen gas mixture and placed on the scanner bed;anesthesia was maintained using 1% isoflurane/gas mixture. Static scanswere recorded at various time points after injection with a minimum of30 million coincident events (10-30 min total scan time). Activityconcentrations (percentage of dose per gram of tissue [% ID/g]) andmaximum intensity projections were determined by conversion of thecounting rates from the reconstructed images. All of the resulting PETimages were analyzed using ASIPro VM™ software. The coronal slices aboveare representative images chosen to illustrate the areas of highestuptake.

FIG. 13 shows PET imaging data for ⁶⁴Cu-Tz-PEG7-NOTA in two healthymice. Healthy female athymic nude mice were administered⁶⁴Cu-Tz-PEG₇-NOTA (300-350 μCi in 200 mL 0.9% sterile saline) viaintravenous tail vein injection (t=0). Approximately 5 minutes prior tothe PET images, mice were anesthetized by inhalation of 2% isoflurane(Baxter Healthcare, Deerfield, Ill.)/oxygen gas mixture and placed onthe scanner bed; anesthesia was maintained using 1% isoflurane/gasmixture. Static scans were recorded at various time points afterinjection with a minimum of 30 million coincident events (10-30 mintotal scan time). Activity concentrations (percentage of dose per gramof tissue [% ID/g]) and maximum intensity projections were determined byconversion of the counting rates from the reconstructed images. All ofthe resulting PET images were analyzed using ASIPro VM™ software. Thecoronal slices above are representative images chosen to illustrate theareas of highest uptake.

FIG. 14 shows PET imaging data for ⁶⁴Cu-Tz-SarAr in a healthy mouse.Healthy female athymic nude mice were administered ⁶⁴Cu-Tz-SarAr(300-350 μCi in 200 μL 0.9% sterile saline) via intravenous tail veininjection (t=0). Approximately 5 minutes prior to the PET images, micewere anesthetized by inhalation of 2% isoflurane (Baxter Healthcare,Deerfield, Ill.)/oxygen gas mixture and placed on the scanner bed;anesthesia was maintained using 1% isoflurane/gas mixture. Static scanswere recorded at various time points after injection with a minimum of30 million coincident events (10-30 min total scan time). Activityconcentrations (percentage of dose per gram of tissue [% ID/g]) andmaximum intensity projections were determined by conversion of thecounting rates from the reconstructed images. All of the resulting PETimages were analyzed using ASIPro VM™ software. Maximum intensityprojections (MIPs) are shown for each time point, and the coronal slicesabove are representative images chosen to illustrate the areas ofhighest uptake (i.e. the kidneys, white arrow).

FIG. 15 shows a plot of the clearance of ⁶⁴Cu-Tz-SarAr from the bloodvs. time (red squares). Fitting of the data points to a biexponentialfunction (dotted line) reveals that greater than 99% of the radioligandclears from the blood with a half-life of ˜16 minutes.

FIG. 16 shows pretargeted PET imaging using ⁶⁴Cu-Tz-SarAr and a 24 haccumulation interval. Female athymic nude mice (n=5) bearingsubcutaneous SW1222 (right shoulder) xenografts (100-150 mm³, 9-12 dayspost-inoculation) were administered 100 mg (0.66 nmol) huA33-TCO (in 200mL 0.9% sterile saline) via intravenous tail vein injection. After anaccumulation interval period of 24 h, the same mice were thenadministered ⁶⁴Cu-Tz-SarAr (400-450 μmCi, 0.66-0.77 nmol, in 200 mL 0.9%sterile saline), also via intravenous tail vein injection (t=0). Thecoronal slices (left) intersect the center of the tumor (white arrow).Maximum intensity projections (MIP) at 4, 12, and 24 h post-injectionare also displayed (right), as well as a co-registered PET/CT imagescollected at 24 h post-injection (far right, perspective flipped).

FIG. 17 shows pretargeted PET imaging using ⁶⁴Cu-Tz-SarAr and a 24 haccumulation interval. Female athymic nude mice (n=5) bearingsubcutaneous SW1222 (right shoulder) xenografts (100-150 mm³, 18-21 dayspost-inoculation) were administered 100 mg (0.66 nmol) huA33-TCO (in 200mL 0.9% sterile saline) via intravenous tail vein injection. After anaccumulation interval of 24 h, the same mice were then administered⁶⁴Cu-Tz-SarAr (400-450 μCi in 200 mL 0.9% sterile saline), also viaintravenous tail vein injection (t=0). The specific activity of⁶⁴Cu-Tz-SarAr was adjusted using cold ^(nat)Cu-Tz-SarAr such that themolar ratio of Tz_(injected):huA33_(Injected)=1:1. Static scans wererecorded at various time points after injection with a minimum of 30million coincident events (10-30 min total scan time). Activityconcentrations (percentage of dose per gram of tissue [% ID/g]) andmaximum intensity projections were determined by conversion of thecounting rates from the reconstructed images. All of the resulting PETimages were analyzed using ASIPro VM™ software. The coronal slicesintersect the center of the tumor, and the maximum intensity projection(MIP) displayed was collected at 24 h post-injection.

FIGS. 18A and 18B show pretargeted PET imaging using ⁶⁴Cu-Tz-SarAr and48 h (18A) and 120 h (18B) accumulation intervals. The coronal slices(left) intersect the center of the tumor (white arrow). Maximumintensity projections (MIP) collected at 24 h post-injection are alsodisplayed (right).

FIG. 18C shows activity concentration in the tumor as a function of bothtime post-injection and accumulation interval for pretargeting with⁶⁴Cu-Tz-SarAr.

FIG. 18D shows activity concentration in the blood as a function of bothtime post-injection and accumulation interval for pretargeting with⁶⁴Cu-Tz-SarAr.

FIG. 18E shows tumor-to-blood activity concentration ratios as afunction of both time post-injection and accumulation interval forpretargeting with ⁶⁴Cu-Tz-SarAr.

FIG. 18F shows tumor-to-muscle activity concentration ratios as afunction of both time post-injection and accumulation interval forpretargeting with ⁶⁴Cu-Tz-SarAr.

FIG. 19 shows pretargeted PET imaging using ⁶⁴Cu-Tz-SarAr and a 48 haccumulation interval. Female athymic nude mice (n=5 per radioligand)bearing subcutaneous SW1222 (right shoulder) xenografts (100-150 mm³,18-21 days post-inoculation) were administered 100 mg (0.66 nmol)huA33-TCO (in 200 mL 0.9% sterile saline) via intravenous tail veininjection. After an accumulation interval of 48 h, the same mice werethen administered ⁶⁴Cu-Tz-SarAr (400-450 μCi in 200 mL 0.9% sterilesaline), also via intravenous tail vein injection (t=0). The specificactivity of ⁶⁴Cu-Tz-SarAr was adjusted using cold ^(nat)Cu-Tz-SarAr suchthat the molar ratio of Tz_(injected):huA33_(injected)=1:1. Static scanswere recorded at various time points after injection with a minimum of30 million coincident events (10-30 min total scan time). Activityconcentrations (percentage of dose per gram of tissue [% ID/g]) andmaximum intensity projections were determined by conversion of thecounting rates from the reconstructed images. All of the resulting PETimages were analyzed using ASIPro VM™ software. The coronal slicesintersect the center of the tumor, and the maximum intensity projection(MIP) displayed was collected at 24 h post-injection.

FIG. 20 shows pretargeted PET imaging using ⁶⁴Cu-Tz-SarAr and a 120 haccumulation interval. Female athymic nude mice (n=5) bearingsubcutaneous SW1222 (right shoulder) xenografts (100-150 mm³, 18-21 dayspost-inoculation) were administered 100 mg (0.66 nmol) huA33-TCO (in 200mL 0.9% sterile saline) via intravenous tail vein injection. After anaccumulation interval of 120 h, the same mice were then administered⁶⁴Cu-Tz-SarAr (400-450 μCi in 200 mL 0.9% sterile saline), also viaintravenous tail vein injection (t=0). The specific activity of⁶⁴Cu-Tz-SarAr was adjusted using cold ^(nat)Cu-Tz-SarAr such that themolar ratio of Tz_(injected):huA33_(Injected)=1:1. Static scans wererecorded at various time points after injection with a minimum of 30million coincident events (10-30 min total scan time). Activityconcentrations (percentage of dose per gram of tissue [% ID/g]) andmaximum intensity projections were determined by conversion of thecounting rates from the reconstructed images. All of the resulting PETimages were analyzed using ASIPro VM™ software. The coronal slicesintersect the center of the tumor, and the maximum intensity projection(MIP) displayed was collected at 24 h post-injection.

FIG. 21 shows graphical comparison of salient tumor-to-tissue activityconcentration ratios at 1 h post-injection created using the differentpretargeted PET imaging strategies discussed (see Tables 11, 14, 16, 18,and 19).

FIG. 22 shows graphical comparison of salient tumor-to-tissue activityconcentration ratios at 4 h post-injection created using the differentpretargeted PET imaging strategies discussed (see Tables 11, 14, 16, 18,and 20).

FIG. 23 shows graphical comparison of salient tumor-to-tissue activityconcentration ratios at 12 h post-injection created using the differentpretargeted PET imaging strategies discussed (see Tables 11, 14, 16, 18,and 21).

FIG. 24 shows graphical comparison of salient tumor-to-tissue activityconcentration ratios at 24 h post-injection created using the differentpretargeted PET imaging strategies discussed (see Tables 11, 14, 16, 18,and 22).

FIGS. 25A-25H show autoradiography, histology, and fluorescencemicroscopy of SW1222 colorectal carcinoma xenografts resected afterpretargeted PET imaging using ⁶⁴Cu-Tz-SarAr and an accumulation intervalof 120 h.

FIG. 25A shows hematoxylin and eosin staining

FIG. 25B shows immunofluorescence staining for the huA33 antibody.

FIG. 25C shows autoradiography indicating the localization of⁶⁴Cu-Tz-SarAr.

FIG. 26D shows an overlay of immunofluorescence staining andautoradiography; (E) higher magnification image of the area enclosed bythe black box in FIG. 25A.

FIGS. 25F-25H show higher magnification images of the same areacorresponding to FIGS. 25B-25D, respectively.

FIG. 26 shows pretargeted PET imaging using ⁶⁴Cu-Tz-NOTA. Female athymicnude mice (n=5) bearing subcutaneous SW1222 (right shoulder) xenografts(100-150 mm³, 18-21 days post-inoculation) were administered 100 mg(0.66 nmol) huA33-TCO (in 200 mL 0.9% sterile saline) via intravenoustail vein injection. After an accumulation interval of 24 h, the samemice were then administered ⁶⁴Cu-Tz-NOTA (400-450 μCi in 200 mL 0.9%sterile saline), also via intravenous tail vein injection (t=0). Thespecific activity of ⁶⁴Cu-Tz-NOTA was adjusted using cold^(nat)Cu-Tz-NOTA such that the molar ratio ofTz_(injected):huA33_(injected)=1:1. Static scans were recorded atvarious time points after injection with a minimum of 30 millioncoincident events (10-30 min total scan time). Activity concentrations(percentage of dose per gram of tissue [% ID/g]) and maximum intensityprojections were determined by conversion of the counting rates from thereconstructed images. All of the resulting PET images were analyzedusing ASIPro VM™ software. The coronal slices intersect the center ofthe tumor (solid white arrow), and the maximum intensity projection(MIP) displayed was collected at 24 h post-injection. Note the activitynot only in the SW1222 xenograft but also in the gut of the mouse(dashed white arrow) at 4 h post-injection.

FIG. 27 shows the radiochemical synthesis of the radioligandTz-PEG₁₁-Al¹⁸F-NOTA ([¹⁸F]2). [¹⁸F]2 was obtained in 54-56%radiochemical yields (RCY) (d.c.) and high specifica activities (SAs)(21.4-26.7 GBq/μmol) after a total synthesis time of 108 min.Purification of the crude reaction mixture using a C18-cartridge gave[¹⁸F]2 in purities of greater than 96%.

FIG. 28 shows results of the biodistribution pretargeting CA19.9 with[¹⁸F]2/5B1-TCO. Subcutaneous BxPC3 xenograft bearing mice wereadministered 5B1-TCO (1.33 nmol) 72 h prior to the injection of the¹⁸F-labeled tracer (1.33 nmol, 1.8-2.0 MBq) via the tail vein before themice were euthanized and the organs collected at the appropriate timepoints.

FIG. 29 shows PET images of Tz-PEG₁₁-NOTA-Al¹⁸F/5B1-TCO pretargetingstrategy. Subcutaneous BxPC3 xenograft bearing mice were administered5B1-TCO (1.33 nmol) 72 h prior to the injection of the ¹⁸F-labeledtracer (1.33 nmol, 18-20 MBq) via the tail vein. Transverse (top) andcoronal (middle) planar images intersect the center of the tumors. Themaximum intensity projections (MIPs, bottom) clearly illustrate tumoruptake after 1 h with increasing tumor-to-background ratios over thecourse of the experiment.

FIGS. 30A and 30B show radioactivity (FIG. 30A) and UV (FIG. 30B) tracesof the radio-HPLC analysis of the radioligand Tz-PEG₁₁-Al[¹⁸F]-NOTA.

FIGS. 31A and 31B show radio-TLC diagrams using 90% MeCN in H₂O asmobile phase showing free radioligand (FIG. 31A) and the click reactionproduct after 15 min incubation (FIG. 31B) at room temperature of theradioligand with TCO-modified 5B1. The results indicated that the clickreaction is complete after 15 min (n=3).

FIG. 32 shows biodistribution data of the radioligand obtained fromhealthy nude mice at 1, 2, and 4 h (n=4) after injection of theradioligand [¹⁸F]2.

FIG. 33 shows the blood half-life of the radioligand was calculated byplotting the % ID/g of the collected blood samples for each time point(n=4) against the corresponding collection time points.

DETAILED DESCRIPTION

Throughout the description, where compositions are described as having,including, or comprising specific components, or where methods aredescribed as having, including, or comprising specific steps, it iscontemplated that, additionally, there are compositions of the presentinvention that consist essentially of, or consist of, the recitedcomponents, and that there are methods according to the presentinvention that consist essentially of, or consist of, the recitedprocessing steps.

It should be understood that the order of steps or order for performingcertain action is immaterial so long as the invention remains operable.Moreover, two or more steps or actions may be conducted simultaneously.

The mention herein of any publication, for example, in the Backgroundsection, is not an admission that the publication serves as prior artwith respect to any of the claims presented herein. The Backgroundsection is presented for purposes of clarity and is not meant as adescription of prior art with respect to any claim.

Described herein are Tz/TCO-based pretargeting strategies using (1) a⁶⁴Cu-sarcophagine-based tetrazine radioligand for pretargeted PETimaging with more rapid excretion of the excess radioligand through thebladder and kidneys and (2) an Al[¹⁸F]-NOTA-labeled tetrazineradioligand. These imaging strategies enable delineation of cancer atearlier time points compared to other imaging strategies and furtherdecrease the radiation dose to healthy tissues compared to directlylabeled antibodies.

In certain embodiments, two radioligands ⁶⁴Cu-Tz-PEG₇-NOTA and⁶⁴Cu-Tz-SarAr—based on ⁶⁴Cu-Tz-NOTA were designed to harbor structuralmodifications to alter the pharmacokinetics of ⁶⁴Cu-Tz-NOTA. The in vivoevaluation of these two constructs revealed that ⁶⁴Cu-Tz-PEG₇-NOTA waseliminated via both the gastrointestinal and renal tracts and⁶⁴Cu-Tz-SarAr was cleared through the renal system alone. Moreover,pretargeted PET imaging and biodistribution experiments using huA33-TCO,⁶⁴Cu-Tz-SarAr, and mice bearing human colorectal carcinoma xenograftsrevealed that this approach delineated malignant tissue with hightumor-to-background contrast at only a fraction of the radiation dosecreated by traditional, directly-labeled radioimmunoconjugates. Alteringthe molecular structure of the tetrazine-bearing radioligand effectivelyeliminated background uptake of the radioligand in the gastrointestinaltract.

In certain embodiments, a TCO-bearing immuno-conjugate of theanti-CA19.9 antibody 5B1 and an Al[¹⁸F]-NOTA-labeled tetrazineradioligand were harnessed for the visualization of CA19.9-expressingBxPC3 pancreatic cancer xenografts. Al-based ¹⁸F imaging of smallmolecules, such as tetrazine, has not been previously achieved due tothe decomposition of tetrazine during radiofluorination.Radiofluorination is advantageous over other radiolabeling methodsbecause, in addition to having a shorter half-life, ¹⁸F is more readilyavailable to produce and therefore more convenient to integrate intohospital workflows. Biodistribution and ¹⁸F-PET imaging data demonstratethat this methodology effectively delineates tumor mass with activityconcentrations up to 6.4% ID/g at 4 h after injection of theradioligand.

⁶⁴Cu-Based Tetrazine Radioligands for Pretargeted Imaging

The present disclosure describes the synthesis and characterization ofthe in vivo behavior of two ⁶⁴Cu-labeled tetrazineradioligands—⁶⁴Cu-Tz-PEG₇-NOTA and ⁶⁴Cu-Tz-SarAr—that possess structuralalterations to modulate their pharmacokinetic profiles. It was foundthat the ⁶⁴Cu-Tz-SarAr radioligand, in combination with the huA33-TCO,performed better than the ⁶⁴Cu-Tz-NOTA radioligand. For example, thecombined huA33-TCO and ⁶⁴Cu-Tz-SarAr system exhibited higher activityconcentrations in the tumor, the ⁶⁴Cu-Tz-SarAr radioligand was rapidlycleared through the renal system allowing for higher tumor-to-backgroundactivity concentration ratios at early time points, and the presentlydescribed system demonstrated dosimetric improvement over otherradioimmunoconjugate systems.

System Design

At a basic level, the pretargeting strategy described herein comprisesthree components: (1) the antibody, (2) the transcyclooctene, and (3)the tetrazine; each component is important. The present disclosuredescribes a pretargeted PET imaging system for colorectal system, whichuses the huA33 antibody. The huA33 antibody is a humanized antibody thattargets the A33 antigen, a transmembrane glycoprotein abundantlyexpressed by greater than 95% of all colorectal cancer tumors. While lowlevels of expression of the A33 antigen have been found on normal bowelepithelium, clinical studies with ¹²⁴I-labeled huA33 have illustratedthat tumor tissue retains the antibody far longer than healthyepithelium. Furthermore, in vitro studies have shown that the huA33-A33antigen complex persists on the surface of the cell for days afterformation. These features are important for pretargeting methodologiesbecause the antibody must accumulate in tumor tissue compared to normaltissue and remain accessible to the radioligand for the in vivo ligationto occur.

The pretargeted PET system includes: (1) injection of the huA33-TCOconjugate; (2) accumulation of the antibody at the target site andallowing unbound antibody to clear from the blood; (3) injection of theradioligand; and (4) an in vivo click ligation of the two components,followed by clearance of the excess radioligand (FIG. 2). Importantly,this pretargeting strategy effectively delineates tumor tissue at muchearlier time points than directly radiolabeled antibodies andsignificantly reduces the overall radiation burden to the patient,permitting safer and more accurate diagnoses in shorter time frames.

The present disclosure also describes use of transcyclooct-4-en-1-ylhydrogen carbonate (TCO) and 3-4-(benzylamine)-1,2,4,5-tetrazine (Tz) asthe transcyclooctene and tetrazine components. The IEDDA cycloadditionbetween these two moieties has been shown to be extraordinarily rapid,with a second order rate constant greater than 30,000M⁻¹ s⁻¹.Furthermore, both components have been shown to be sufficiently stablein physiological settings, and amine-reactive variants (TCO-NHS andTz-NHS) of both are commercially available.

As described above, the relatively slow (t_(1/2)˜4 h) clearance of the⁶⁴Cu-Tz-NOTA (FIG. 3A) through the intestinal tract has hamperedpotential use as a methodology for imaging colorectal cancer. Withoutbeing bound by theory, it is thought that PEG linkers—also known asoligoethyleneglycol linkers—can accelerate the clearance and lower thenon-target tissue uptake of radiopharmaceuticals. Likewise, withoutbeing bound by theory, it is thought that changes to the identity of thechelator and the overall charge of the radiometal-chelator complex candramatically influence pharmacokinetics. Thus, two ⁶⁴Cu-Tz radioligandswith structural alterations made to ⁶⁴Cu-Tz-NOTA were designed forimproved pharmacokinetic profiles. To this end, two different structuralmotifs (e.g., polyethyleneglycol (PEG) linkers and chelators) were usedto influence the in vivo behavior of radiotracers.

In a first example disclosed herein, ⁶⁴Cu-Tz-PEG₇-NOTA contained a PEG₇spacer that separated the tetrazine moiety from the NOTA chelator (FIG.3B). In a second example disclosed herein, ⁶⁴Cu-Tz-SarAr contained asarcophagine-based chelator (SarAr) that replaced the NOTA macrocycle.This chelator substitution not only changed the coordination environmentfrom N₃O₃ to N₆ but shifted the overall charge of the metal-ligandcomplex from −1 (Cu^(II)-NOTA) to +2 (Cu^(II)-SarAr) (FIG. 3C). Table 0depicts some selected properties of the three tetrazine radioligandsshown in FIGS. 3A-3C.

TABLE 0 % Intact % Intact Molecular After 2 h After 2 h weightCoordination Net @ 37° C. @ 37° C. Radioligand (MW) Environment ChargeLog D (PBS) (Serum) ⁶⁴Cu-Tz- 752.6 N₃O₃ −1 −2.54 +/− 0.10 91.6 +/− 1.985.4 +/− 6.6 NOTA ⁶⁴Cu-Tz- 1163.2 N₃O₃ −1 −2.44 +/− 0.08 87.0 +/− 1.384.7 +/− 4.8 PEG₇-NOTA ⁶⁴Cu-Tz- 780.9 N₆ +2 −2.08 +/− 0.06 94.7 +/− 0.692.0 +/− 2.1 SarAr

Synthesis and Characterization

Tz-PEG₇-NOTA was synthesized in 49% yield over three facile steps: thecoupling of Tz-NHS and monofunctionalO-(2-aminoethyl)-O′-[2-(bocamino)ethyl]hexaethylene glycol to formTz-PEG₇-NHBoc; the removal of the tert-butyloxycarbonyl protecting groupwith TFA/CH₂Cl₂; and the coupling of Tz-PEG₇-NH₂ with p-NCS-Bn-NOTA(FIG. 4). Given the symmetry of its sarcophagine precursor, Tz-SarArrequired more complex synthesis. In this case, the mono-alkylation ofDiAmSar with Boc-protected 4-(bromomethyl)-benzylamine was followed bythe removal of the acid-labile protecting group with TFA and thecoupling of the resulting SarAr-Bn-NH₂ moiety with Tz-NHS to produce thefinal product in 29% yield over three steps (FIG. 5). For bothsyntheses, all intermediates as well as the completed tetrazine-bearingprecursors were analyzed and purified using reverse-phase C₁₈ HPLC andcharacterized via UV-Vis spectrophotometry, ¹H-NMR, ESI-MS, andhigh-resolution mass spectrometry.

Once the precursors were made, the tetrazine constructs were thenradiolabeled via incubation with [⁶⁴Cu]—CuCl₂ for 10 minutes at roomtemperature in 200 mM NH₄OAc, pH 5.0 and purified via reverse-phase C₁₈HPLC (t_(R)=8.7 min for ⁶⁴Cu-Tz-SarAr and 9.7 min for ⁶⁴Cu-Tz-PEG₇NOTA;FIGS. 6-9). In both cases, the identity of the radiolabeled product wasconfirmed via co-injection of unlabeled ^(nat)Cu-Tz-SarAr and^(nat)Cu-Tz-PEG_(S)-NOTA standards. Ultimately, ⁶⁴Cu-Tz-PEG₇NOTA wasprepared in greater than 99% radionuclidic purity, 78±6% decay-correctedisolated yield, and a specific activity of 323±37 mCi/μmol (n=6).Similarly, ⁶⁴Cu-Tz-SarAr was synthesized in greater than 99%radionuclidic purity, 79±7% decay-corrected isolated yield, and aspecific activity of 310±36 mCi/μmol (n=6). The ⁶⁴Cu-Tz-NOTA radioligandwas synthesized, radiolabeled, and purified as previously reported byZeflis et al. J. Nucl. Med. 2013, 54, 1389-1396, which is herebyincorporated by reference in its entirety. In order to probe theinfluence of the structural changes on solubility, the partitioncoefficients of the various radioligands were determined using PBS (pH7.4) and 1-octanol (Table 1). Table 1 shows partition coefficient (LogD) of the ⁶⁴Cu-labeled tetrazines in 1-octanol and PBS (pH 7.4). Whileall three radioligands proved reasonably hydrophilic (e.g., log D valuesbelow −2), differences were observed. For example, the replacement ofNOTA with SarAr rendered ⁶⁴Cu-Tz-SarAr (log D=−2.08±0.06) morehydrophobic than ⁶⁴Cu-Tz-NOTA (log D=−2.54±0.1). Moreover, the additionof the PEG₇ moiety in ⁶⁴Cu-Tz-PEG₇-NOTA did not generate a morehydrophilic product (log D=−2.44±0.08) than the ⁶⁴Cu-Tz-NOTA radioligandthat lacked a linker.

TABLE 1 Radioligand LogD ⁶⁴Cu-Tz-NOTA −2.54 ± 0.10 ⁶⁴Cu-Tz-PEG₇-NOTA−2.44 ± 0.08 ⁶⁴Cu-Tz-SarAr −2.08 ± 0.06

Next, the aqueous and serum stabilities were determined via incubationat 37° C. (Tables 2-3). Both ⁶⁴Cu-Tz-PEG₇-NOTA and ⁶⁴Cu-Tz-SarAr wereshown to be fairly stable in PBS (pH 7.4). It was determined that94.7±0.6% and 93.3±0.5% of ⁶⁴Cu-Tz-SarAr remained intact after 2 and 4h, respectively. ⁶⁴Cu-Tz-PEG₇-NOTA proved similarly stable, with87.0±1.3% (2 h) and 83.2±4.6% (4 h) intact. However, more extensivedecomposition was observed in human serum. For example, after a 4 hincubation, 77.8±3.5% and 81.2±3.7% of ⁶⁴Cu-Tz-PEG_(S)-NOTA and⁶⁴Cu-Tz-SarAr, respectively, remained intact. Importantly, the releaseof ⁶⁴CU²⁺ from the chelators was not observed in any of the trials.Despite the rates of decomposition, the speed of the IEDDA reaction wasfaster than short blood half-lives of these small molecules; therefore,the radioligands remained functional in vivo. Table 2 shows the percentof ⁶⁴Cu-Tz-PEG₇-NOTA and ⁶⁴Cu-Tz-SarAr intact after incubation in PBS(pH 7.4) at 37° C. Table 3 shows the percent of ⁶⁴Cu-Tz-PEG₇-NOTA and⁶⁴Cu-Tz-SarAr intact after incubation in human serum at 37° C.

TABLE 2 ⁶⁴Cu-Tz-PEG₇- Time NOTA ⁶⁴Cu-Tz-SarAr 2 h 87.0 ± 1.3 94.7 ± 0.64 h 83.2 ± 4.6 93.3 ± 0.5 8 h 76.2 ± 2.6 89.7 ± 2.3

TABLE 3 ⁶⁴Cu-Tz-PEG₇- Time NOTA ⁶⁴Cu-Tz-SarAr 2 h 84.7 ± 4.8 92.0 ± 2.14 h 77.8 ± 3.5 81.2 ± 3.7 8 h 64.0 ± 6.5 67.7 ± 4.3

Next, huA33-TCO was synthesized via the coupling of TCO-NHS to the huA33antibody as previously described (FIG. 10). Briefly, a solution of huA33(2-3 mg/mL) in PBS was adjusted to pH 8.8-9.0 with 0.1 M Na₂CO₃. Tenmolar equivalents of TCO-NHS were then added to the antibody solution,and the resulting reaction mixture was incubated at room temperature for1 hr prior to purification via size exclusion chromatography. Using afluorophore-labeled tetrazine probe (Tz-PEG₇-AF680), the TCO occupancyof the huA33 was determined to be 3.6±0.6 TCO/mAb. Both⁶⁴Cu-Tz-PEG₇-NOTA and ⁶⁴Cu-Tz-SarAr were incubated with huA33-TCO andrapidly resulted in greater than 95% reaction yields. Afterpurification, ⁶⁴Cu-labeled huA33 radioimmunoconjugates exhibited highspecific activities (greater than 2 mCi/mg) and immunoreactive fractions(greater than 0.95) with A33 antigen-expressing SW1222 human colorectalcarcinoma cells (Table 4). Control reactions between unmodified huA33and the ⁶⁴Cu-labeled tetrazines as well as huA33-TCO and uncomplexed⁶⁴CU²⁺ resulted in less than 1% radiolabeling of the huA33 constructs.Table 4 shows immunoreactive fractions of radioimmunoconjugates formedthrough the reaction of huA33-TCO and either ⁶⁴Cu-Tz-PEG₇-NOTA or⁶⁴Cu-Tz-SarAr, as determined via in vitro assays with A33antigen-expressing SW1222 cells.

TABLE 4 Immunoreactive Radioimmunoconjugate Fraction huA33-TCO +⁶⁴Cu-Tz-PEG₇- 0.96 ± 0.02 NOTA huA33-TCO + ⁶⁴Cu-Tz-SarAr 0.97 ± 0.01

In Vivo Evaluation of the ⁶⁴ Cu-Labeled Tetrazines

After synthesis and characterization, in vivo behavior andpharmacokinetics of the ⁶⁴Cu-labeled tetrazine radioligands weremeasured. To this end, acute biodistribution and PET imaging experimentswere performed in healthy athymic nude mice injected with ⁶⁴Cu-Tz-NOTA,⁶⁴Cu-Tz-PEG₇-NOTA, or ⁶⁴Cu-Tz-SarAr (FIGS. 12-14 and Tables 5-7). Allthree ⁶⁴Cu-labeled tetrazines minimally accumulated in most healthynon-target tissues, with activity concentrations lower than 0.5% ID/gbeyond the earliest time points. Notably, the largest difference betweenthe three radioligands was their excretion profiles. As discussedpreviously, ⁶⁴Cu-Tz-NOTA was excreted relatively slowly through thefeces, with 10.0±1.3% ID/g remaining in the large intestine and itscontents at 4 h post-injection, a number which dropped to 1.4±0.7% ID/gat 24 h. The addition of the PEG linker in ⁶⁴Cu-Tz-PEG₇-NOTA caused twodistinct changes. First, the elimination of the radioligand through thegut was accelerated, with only 4.9±0.7% ID/g in the large intestine andits contents at 4 h post-injection Second, the kidneys displayedactivity concentrations of 1.5±0.2% ID/g and 1.2±0.4% ID/g at 1 h and 4h, respectively, indicating that a portion of the radiotracer wasexcreted by the urinary tract. Notably, the largest difference wasobserved with ⁶⁴Cu-Tz-SarAr, which was excreted exclusively and rapidlythrough the renal system. The tissue with the highest levels of⁶⁴Cu-Tz-SarAr at 1 h post-injection was the kidney, with 2.3±0.4% ID/g.The activity in the kidneys decreased over time; however, at 24 h someretention of the radioligand remained. Complementary PET imagingexperiments confirmed the observations from the biodistributions:⁶⁴Cu-Tz-SarAr cleared quickly and cleanly through the urinary tract;⁶⁴Cu-Tz-NOTA was eliminated somewhat sluggishly through thegastrointestinal pathway; and ⁶⁴Cu-Tz-PEG₇-NOTA represented anintermediate case, with excretion through both the intestines and thekidneys.

Table 5 shows biodistribution data (% ID/g±SD) of ⁶⁴Cu-Tz-NOTA versustime in healthy athymic nude (n=4 for each time point). Mice wereadministered ⁶⁴Cu-Tz-NOTA (25-30 μCi in 200 μL, 0.9% sterile saline) viaintravenous tail vein injection. Animals (n=4 per group) were euthanizedby CO₂(g) asphyxiation at 1, 4, and 24 h after injection. Afterasphyxiation, tissues were removed, rinsed in water, dried in air for 5min, weighed, and counted in a gamma counter calibrated for ⁶⁴Cu. Countswere converted into activity using a calibration curve generated fromknown standards. Count data were background- and decay-corrected to thetime of injection, and the percent injected dose per gram (% ID/g) foreach tissue sample was calculated by normalization to the total activityinjected. Contents within the stomach, small intestine, and largeintestine were not removed for the measurements.

TABLE 5 1 h 4 h 24 h Blood 0.23 ± 0.01 0.13 ± 0.01 0.07 ± 0.01 Heart0.10 ± 0.02 0.11 ± 0.02 0.09 ± 0.01 Lung 0.38 ± 0.06 0.27 ± 0.08 0.21 ±0.02 Liver 1.45 ± 0.39 0.42 ± 0.13 0.21 ± 0.06 Spleen 0.15 ± 0.05 0.09 ±0.02 0.08 ± 0.02 Stomach 0.12 ± 0.04 0.05 ± 0.03 0.18 ± 0.11 LargeIntestine 11.58 ± 2.19  10.03 ± 1.33  1.37 ± 0.65 Small Intestine 3.14 ±1.47 0.17 ± 0.07 0.10 ± 0.02 Kidney 0.53 ± 0.09 0.33 ± 0.07 0.22 ± 0.07Muscle 0.04 ± 0.02 0.03 ± 0.02 0.03 ± 0.03 Bone 0.10 ± 0.03 0.07 ± 0.040.03 ± 0.01

Table 6 shows biodistribution data (% ID/g±SD) of ⁶⁴Cu-Tz-PEG₇-NOTAversus time in healthy athymic nude (n=4 for each time point). Mice wereadministered ⁶⁴Cu-Tz-PEG₇-NOTA (25-30 μCi in 200 μL, 0.9% sterilesaline) via intravenous tail vein injection. Animals (n=4 per group)were euthanized by CO₂(g) asphyxiation at 1, 4, and 24 h afterinjection. After asphyxiation, tissues were removed, rinsed in water,dried in air for 5 min, weighed, and counted in a gamma countercalibrated for ⁶⁴Cu. Counts were converted into activity using acalibration curve generated from known standards. Count data werebackground- and decay-corrected to the time of injection, and thepercent injected dose per gram (% ID/g) for each tissue sample wascalculated by normalization to the total activity injected. Contentswithin the stomach, small intestine, and large intestine were notremoved for the measurements.

TABLE 6 1 h 4 h 24 h Blood 0.38 ± 0.14 0.26 ± 0.14 0.08 ± 0.01 Heart0.17 ± 0.02 0.18 ± 0.03 0.09 ± 0.01 Lung 0.49 ± 0.11 0.35 ± 0.13 0.17 ±0.03 Liver 0.77 ± 0.11 0.63 ± 0.2  0.32 ± 0.11 Spleen 0.19 ± 0.01 0.14 ±0.02 0.12 ± 0.01 Stomach 0.18 ± 0.06 0.36 ± 0.25 0.09 ± 0.02 LargeIntestine 6.35 ± 0.69 4.91 ± 0.71 0.40 ± 0.15 Small Intestine  0.5 ±0.26  0.3 ± 0.14 0.10 ± 0.01 Kidney 1.51 ± 0.24 1.23 ± 0.39 0.86 ± 0.11Muscle 0.05 ± 0.01 0.03 ± 0.02 0.02 ± 0.01 Bone 0.09 ± 0.06 0.07 ± 0.020.06 ± 0.01

Table 7 shows biodistribution data (% ID/g±SD) of ⁶⁴Cu-Tz-SarAr versustime in healthy athymic nude (n=4 for each time point). Mice wereadministered ⁶⁴Cu-Tz-SarAr (25-30 μCi in 200 μL 0.9% sterile saline) viaintravenous tail vein injection. Animals (n=4 per group) were euthanizedby CO₂(g) asphyxiation at 1, 4, and 24 h after injection. Afterasphyxiation, tissues were removed, rinsed in water, dried in air for 5min, weighed, and counted in a gamma counter calibrated for ⁶⁴Cu. Countswere converted into activity using a calibration curve generated fromknown standards. Count data were background- and decay-corrected to thetime of injection, and the percent injected dose per gram (% ID/g) foreach tissue sample was calculated by normalization to the total activityinjected. Contents within the stomach, small intestine, and largeintestine were not removed for the measurements.

TABLE 7 1 h 4 h 24 h Blood 0.42 ± 0.11 0.15 ± 0.01 0.06 ± 0.01 Heart0.22 ± 0.06 0.12 ± 0.02 0.08 ± 0.02 Lung 0.32 ± 0.06 0.31 ± 0.09 0.24 ±0.06 Liver 0.72 ± 0.03 0.42 ± 0.24 0.28 ± 0.07 Spleen 0.38 ± 0.06  0.2 ±0.05 0.21 ± 0.04 Stomach 0.24 ± 0.14 0.2 ± 0.1 0.05 ± 0.01 LargeIntestine 0.07 ± 0.01 0.08 ± 0.03 0.07 ± 0.01 Small Intestine 0.29 ±0.21 0.16 ± 0.03 0.07 ± 0.01 Kidney 2.34 ± 0.35 1.57 ± 0.19 1.26 ± 0.41Muscle 0.05 ± 0.03 0.04 ± 0.01 0.03 ± 0.01 Bone 0.12 ± 0.03 0.09 ± 0.010.08 ± 0.02

Thus, ⁶⁴Cu-Tz-SarAr possessed improved elimination pharmacokineticscompared to other pretargeted PET strategies. The radioligand wasexcreted rapidly via the bladder and kidneys, and the activityconcentrations in the large intestine—a critical source of backgroundnoise in clinical colorectal cancer imaging—remained remarkably low(e.g., 0.07±0.01% ID/g at 1 h post-injection). Additional experimentswere conducted to further interrogate the in vivo performance of⁶⁴Cu-Tz-SarAr. Blood activity measurements, for example, revealed thatthe vast majority of the radioligand cleared from the blood with aresidence time of ˜16 minutes (see Table 8 and FIG. 15). Moreover, invivo stability assays indicated that 96.8±0.8% of the ⁶⁴Cu-Tz-SarArremained intact at 15 minutes post-injection, a value which falls to82.0±3.3% at 1 h post-injection and ultimately 29.0±5.9% 4 h afteradministration (see Table 9). These numbers clearly indicated that while⁶⁴Cu-Tz-SarAr was not tremendously stable in vivo, the radioligandunquestionably remained intact during the critical initial bloodresidence time frame.

Table 8 shows the in vivo blood residence time of ⁶⁴Cu-Tz-SarAr inhealthy athymic nude mice (n=3).

TABLE 8 Time Activity in Blood (% ID/g) 15 min 3.02 ± 1.10 30 min 1.55 ±0.21  1 h 0.42 ± 0.11  4 h 0.15 ± 0.01 12 h 0.12 ± 0.03 24 h 0.06 ± 0.01

Table 9 shows percent of ⁶⁴Cu-Tz-SarAr intact in blood after vs. time.

TABLE 9 % Intact ⁶⁴Cu-Tz- Time SarAr 15 min 96.8 ± 0.8  1 h 82.0 ± 3.3 4 h 29.0 ± 5.9

In Vivo Pretargeting

Due to its pharmacokinetic profile, ⁶⁴Cu-Tz-SarAr was selected for invivo pretargeting experiments. To this end, athymic nude mice bearingA33 antigen-expressing SW1222 human colorectal carcinoma tumors werefirst injected with huA33-TCO (100 μg). After a 24 h interval duringwhich huA33-TCO accumulated at the tumor and cleared from the blood, themice were injected with ⁶⁴Cu-Tz-SarAr (400-450 μCi). The specificactivity of the radiotracer was adjusted using cold ^(nat)Cu-Tz-SarArsuch that the molar ratio of Tz-SarAr:huA33-TCO≈1:1. Both the PETimaging (FIG. 16 and FIG. 17) and biodistribution results (Tables 10-12)indicated that the strategy quickly and clearly delineated colorectalcarcinoma tissue with low activity concentrations in non-target tissues.At 1 h post-injection, the activity concentration in the tumor(5.63±0.67% ID/g) was the highest of all tissues surveyed, while theblood (4.2±0.8% ID/g) and kidneys (3.1±0.3% ID/g) were the healthyorgans with the highest background activity concentrations. Notably,over the course of the experiment, the activity in the non-targettissues cleared. For example, at 24 h post-injection, the activityconcentration in the blood and kidneys was reduced to 2.2±0.4% ID/g and1.9±0.4% ID/g, respectively, while the tumoral activity concentration atthe same time point was 6.7±1.3% ID/g. Furthermore, all other non-targettissues contained less than 1.5% ID/g at these later time points.Moreover, the tumor-to-background activity ratios were favorable atearly time points (e.g., tumor:muscle=14.9±2.9 at 1 h post-injection)and increased to 45.12±8.5 at 12 h and 37.37±16.1 at 24 h (Table 11).Control PET imaging experiments were run using ⁶⁴Cu-Tz-SarAr alone aswell as ⁶⁴Cu-Tz-SarAr with unmodified huA33. As expected, both casesresulted in minimal (less than 0.5% ID/g) uptake by the tumor.

Table 10 shows the biodistribution of ⁶⁴Cu-Tz-SarAr pretargetingexperiment with a 24 h accumulation interval. Female athymic nude micebearing subcutaneous SW1222 (right shoulder) xenografts (100-150 mm³,18-21 days post-inoculation) were administered 100 μg (0.66 nmol)huA33-TCO (in 200 μL, 0.9% sterile saline) via intravenous tail veininjection. After an accumulation interval of 24 h, the same mice werethen administered ⁶⁴Cu-Tz-SarAr (300-350 μCi in 200 μL, 0.9% sterilesaline), also via intravenous tail vein injection (t=0). The specificactivity of the radiotracer was adjusted using cold ^(nat)Cu-Tz-SarArsuch that the molar ratio of Tz_(injected):huA33_(Injected)=1:1. Animals(n=4 per group) were euthanized by CO₂(g) asphyxiation at 1, 4, 12, and24 h after injection. After asphyxiation, 13 tissues were removed,rinsed in water, dried in air for 5 min, weighed, and counted in a gammacounter calibrated for ⁶⁴Cu. Counts were converted into activity using acalibration curve generated from known standards. Count data werebackground- and decay-corrected to the time of injection, and thepercent injected dose per gram (% ID/g) for each tissue sample wascalculated by normalization to the total activity injected. Contentswithin the stomach, small intestine, and large intestine were notremoved for the measurements.

TABLE 10 1 h 4 h 12 h 24 h Blood 4.20 ± 0.80 4.00 ± 0.37 2.19 ± 0.392.61 ± 0.20 Tumor 5.63 ± 0.67 5.56 ± 0.91 6.74 ± 1.26 7.38 ± 2.02 Heart1.81 ± 0.46 1.45 ± 0.03 0.84 ± 0.1 0.81 ± 0.04 Lung 1.65 ± 0.51 1.55 ±0.45 1.24 ± 0.22 0.99 ± 0.24 Liver 1.60 ± 0.12 1.45 ± 0.21 1.33 ± 0.531.51 ± 0.2 Spleen 0.98 ± 0.25 0.81 ± 0.20 0.65 ± 0.17 0.64 ± 0.04Stomach 0.73 ± 0.19 0.56 ± 0.20 0.21 ± 0.1 0.28 ± 0.07 Large Intestine0.21 ± 0.09 0.48 ± 0.09 0.20 ± 0.04 0.26 ± 0.06 Small Intestine 0.88 ±0.09 0.62 ± 0.06 0.35 ± 0.08 0.45 ± 0.07 Kidney 3.08 ± 0.28 2.77 ± 0.571.87 ± 0.42 2.00 ± 0.24 Muscle 0.38 ± 0.06 0.37 ± 0.07 0.15 ± 0.01 0.20± 0.07 Bone 0.67 ± 0.14 0.34 ± 0.18 0.23 ± 0.05 0.29 ± 0.05

Table 11 shows tumor-to-tissue activity concentration ratios derivedfrom the ⁶⁴Cu-Tz-SarAr pretargeting biodistribution experiment with a 24h accumulation interval as shown in Table 10.

TABLE 11 1 h 4 h 12 h 24 h Tumor/Blood 1.34 ± 0.30 1.39 ± 0.26 3.07 ±0.79 2.83 ± 0.81 Tumor/Heart 3.11 ± 0.87 3.84 ± 0.63 8.01 ± 1.77 9.10 ±2.54 Tumor/Lung 3.40 ± 1.12 3.59 ± 1.20 5.43 ± 1.41 7.44 ± 2.74Tumor/Liver 3.52 ± 0.49 3.83 ± 0.83 5.06 ± 2.23 4.87 ± 1.48 Tumor/Spleen5.75 ± 1.61 6.88 ± 2.01 10.34 ± 3.27  11.45 ± 3.20  Tumor/Stomach 7.75 ±2.24 9.91 ± 3.85 31.76 ± 16.62 26.44 ± 9.89  Tumor/Blood 1.34 ± 0.301.39 ± 0.26 3.07 ± 0.79 2.83 ± 0.81 Tumor/Large Intestine 26.88 ± 12.1111.65 ± 2.99  33.67 ± 8.82  28.86 ± 10.28 Tumor/Small Intestine 6.42 ±1.01 8.96 ± 1.71 19.37 ± 5.95  16.33 ± 5.12  Tumor/Kidney 1.83 ± 0.272.00 ± 0.53 3.61 ± 1.05 3.68 ± 1.10 Tumor/Muscle 14.92 ± 2.85  14.84 ±3.69  45.12 ± 8.55  37.37 ± 16.06 Tumor/Bone 8.43 ± 2.07 16.15 ± 8.89 29.8 ± 8.38 25.46 ± 8.22 

Table 12 shows biodistribution data for in vivo pretargeting experimentusing ⁶⁴Cu-Tz-SarAr and a 24 h accumulation interval.

TABLE 12 1 h 4 h 12 h 24 h Blood 4.20 ± 0.80^(a) 4.00 ± 0.37 2.19 ± 0.392.61 ± 0.20 Tumor 5.63 ± 0.67 5.56 ± 0.91 6.74 ± 1.26 7.38 ± 2.02 Heart1.81 ± 0.46 1.45 ± 0.03 0.84 ± 0.10 0.81 ± 0.04 Lung 1.65 ± 0.51 1.55 ±0.45 1.24 ± 0.22 0.99 ± 0.24 Liver 1.60 ± 0.12 1.45 ± 0.21 1.33 ± 0.531.51 ± 0.20 Spleen 0.98 ± 0.25 0.81 ± 0.20 0.65 ± 0.17 0.64 ± 0.04Stomach 0.73 ± 0.19 0.56 ± 0.20 0.21 ± 0.10 0.28 ± 0.07 Large Intestine0.21 ± 0.09 0.48 ± 0.09 0.20 ± 0.04 0.26 ± 0.06 Small Intestine 0.88 ±0.09 0.62 ± 0.06 0.35 ± 0.08 0.45 ± 0.07 Kidney 3.08 ± 0.28 2.77 ± 0.571.87 ± 0.42 2.00 ± 0.24 Muscle 0.38 ± 0.06 0.37 ± 0.07 0.15 ± 0.01 0.20± 0.07 Bone 0.67 ± 0.14 0.34 ± 0.18 0.23 ± 0.05 0.29 ± 0.05 ^(a)Valuesare % ID/g ± SD. Mice (n = 4) bearing subcutaneous SW1222 xenograftswere administered huA33-TCO via tail vein injection. After 24 h, thesame mice were administered ⁶⁴Cu-Tz-SarAr, also via tail vein injection.

Moreover, the PET imaging and biodistribution data for both designedsystems outperformed the ⁶⁴Cu-Tz-NOTA system (FIG. 26 and Tables 13-14).First, the activity concentrations in the tumor were higher using the⁶⁴Cu-Tz-SarAr approach. For example, ⁶⁴Cu-Tz-SarAr demonstrated anactivity concentration of 5.56±0.91% ID/g in the tumor whereas⁶⁴Cu-Tz-NOTA demonstrated an activity concentration of 4.09±0.61% ID/gat the same time-point of 4 h post-injection Second, the chelatorstructure alteration disclosed herein achieved highertumor-to-background activity ratios compared to the ⁶⁴Cu-Tz-NOTAradioligand. For example, at 12 h after injection, the tumor-to-muscleand tumor-to-blood activity ratios for ⁶⁴Cu-Tz-SarAr were 3.1±0.8 and45.1±8.6, respectively, compared to 1.8±0.5 and 26.6±6.6 for⁶⁴Cu-Tz-NOTA. Third, the clearance of the ⁶⁴Cu-Tz-SarAr and ⁶⁴Cu-Tz-NOTAradiotracers through the GI tract differed. For example, at 1 hpost-injection, the activity concentration of ⁶⁴Cu-Tz-NOTA in the largeintestine and its contents was 13.29±3.15% ID/g compared to an activityconcentration of 0.21±0.09% ID/g for ⁶⁴Cu-Tz-SarAr. This caused thetumor-to-large intestine activity ratio to be 1.44±0.72 for ⁶⁴Cu-Tz-NOTAto 33.67±8.82 for ⁶⁴Cu-Tz-SarAr at 12 h post-injection However, the⁶⁴Cu-Tz-SarAr system exhibited higher activity concentrations in itsclearance organs, the kidneys. Still, the activity levels of⁶⁴Cu-Tz-SarAr in the kidneys were generally below activity levels of⁶⁴Cu-Tz-NOTA measured in the gut. It is important to note that lowlevels of residual uptake in the kidneys will not significantlyinterfere with clinical imaging of primary colorectal carcinoma unlikeuptake in the large intestine.

Table 13 shows the biodistribution of ⁶⁴Cu-Tz-NOTA with a 24 haccumulation interval. Contents within the stomach, small intestine, andlarge intestine were not removed for the measurements. This data wasoriginally published in JNM. Zeglis, B. M. et al. “A pretargeted PETimaging strategy based on bioorthogonal Diels-Alder click chemistry.”Journal of Nuclear Medicine. 54, 1389-1396 (2013). ©2013 by the Societyof Nuclear Medicine and Molecular Imaging, Inc.

TABLE 13 1 h 4 h 12 h 24 h Blood 3.47 ± 0.63 2.61 ± 0.83 2.31 ± 0.4 2.07 ± 0.49 Tumor 4.07 ± 0.25 4.09 ± 0.61 4.20 ± 0.84 3.94 ± 0.92 Heart1.09 ± 0.18 0.91 ± 0.27 0.92 ± 0.14 0.82 ± 0.21 Lung 1.58 ± 0.46  1.6 ±0.39 1.09 ± 0.38 1.03 ± 0.31 Liver 2.19 ± 0.25 1.26 ± 0.3  0.93 ± 0.231.07 ± 0.15 Spleen 0.63 ± 0.07 0.51 ± 0.22 0.59 ± 0.25 0.45 ± 0.11Stomach 0.45 ± 0.08 0.25 ± 0.12 0.52 ± 0.64 0.16 ± 0.03 Large 13.29 ±3.15  9.43 ± 4.22 2.92 ± 1.34 1.67 ± 0.88 Intestine Blood 3.47 ± 0.632.61 ± 0.83 2.31 ± 0.4  2.07 ± 0.49 Small 0.03 ± 0.04 0.38 ± 0.08 0.77 ±0.51 0.35 ± 0.04 Intestine Kidney  1.3 ± 0.15 0.95 ± 0.31 0.91 ± 0.29 0.7 ± 0.19 Muscle 0.22 ± 0.04 0.14 ± 0.03 0.16 ± 0.02 0.15 ± 0.02 Bone 0.3 ± 0.16 0.27 ± 0.24 0.35 ± 0.11 0.29 ± 0.07

Table 14 shows tumor-to-tissue activity concentration ratios of⁶⁴Cu-Tz-NOTA pretargeting experiment with a 24 h accumulation interval.This data was originally published in JNM. Zeglis, B. M. et al. “Apretargeted PET imaging strategy based on bioorthogonal Diels-Alderclick chemistry.” Journal of Nuclear Medicine. 54, 1389-1396 (2013).©2013 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

TABLE 14 1 h 4 h 12 h 24 h Tumor/Blood 1.17 ± 0.22 1.57 ± 0.55 1.82 ±0.48  1.9 ± 0.63 Tumor/Heart 3.75 ± 0.65 4.51 ± 1.49 4.58 ± 1.16 4.82 ±1.69 Tumor/Lung 2.57 ± 0.76 2.55 ± 0.73 3.87 ± 1.55 3.83 ± 1.45Tumor/Liver 1.86 ± 0.24 3.23 ± 0.9  4.53 ± 1.45 3.69 ± 1.01 Tumor/Spleen6.46 ± 0.81 7.97 ± 3.57  7.1 ± 3.34 8.79 ± 2.98 Tumor/Stomach 8.96 ±1.7  16.17 ± 8.21   8.12 ± 10.12 24.29 ± 6.89  Tumor/Large Intestine0.31 ± 0.08 0.43 ± 0.2  1.44 ± 0.72 2.36 ± 1.37 Tumor/Small Intestine119.52 ± 148.82 10.83 ± 2.8  5.46 ± 3.81 11.4 ± 2.91 Tumor/Kidney 3.14 ±0.41 4.32 ± 1.56  4.6 ± 1.71 5.61 ± 1.98 Tumor/Muscle 18.42 ± 3.71 29.38 ± 8.5  26.63 ± 6.59  26.98 ± 7.41  Tumor/Bone 13.72 ± 7.63  14.95± 13.2  11.98 ± 4.51  13.43 ± 4.5 

With a 24 h accumulation interval, the activity concentration of⁶⁴Cu-Tz-SarAr in the tumor reached a relatively high 5.63±0.67% ID/g at1 h post-injection This value increased slightly over the course of theexperiment to 6.74±1.26% ID/g at 12 h and 7.38±2.02% ID/g at 24 h. Thesedata, combined with the relatively high blood activity values, suggestedthat while the majority of the in vivo ligations occurred at the tumor,some radioligands clicked with the antibody in the blood and reached thetumor thereafter. Therefore, in an attempt to decrease the frequency ofclick reactions in the blood and reduce non-target tissue activityconcentrations, PET imaging and biodistribution experiments wereconducted using longer accumulation intervals of 48 and 120 h (FIGS.18A-18B and FIGS. 19-24 and Tables 15-22).

Table 15 shows the biodistribution of ⁶⁴Cu-Tz-SarAr pretargetingexperiment with a 48 h accumulation interval. Female athymic nude micebearing subcutaneous SW1222 (right shoulder) xenografts (100-150 mm³,18-21 days post-inoculation) were administered 100 μg (0.66 nmol)huA33-TCO (in 200 μL 0.9% sterile saline) via intravenous tail veininjection. After an accumulation interval of 48 h, the same mice werethen administered ⁶⁴Cu-Tz-SarAr (300-350 μCi in 200 μL 0.9% sterilesaline) via intravenous tail vein injection (t=0). The specific activityof the radiotracer was adjusted using cold ^(nat)Cu-Tz-SarAr such thatthe molar ratio of Tz_(injected):huA33_(injected)=1:1. Animals (n=4 pergroup) were euthanized by CO₂(g) asphyxiation at 1, 4, 12, and 24 hafter injection. After asphyxiation, tissues were removed, rinsed inwater, dried in air for 5 min, weighed, and counted in a gamma countercalibrated for ⁶⁴Cu. Counts were converted into activity using acalibration curve generated from known standards. Count data werebackground- and decay-corrected to the time of injection, and thepercent injected dose per gram (% ID/g) for each tissue sample wascalculated by normalization to the total activity injected. Contentswithin the stomach, small intestine, and large intestine were notremoved for the measurements.

TABLE 15 1 h 4 h 12 h 24 h Blood 2.15 ± 0.5  1.78 ± 0.28 1.81 ± 0.540.92 ± 0.31 Tumor 4.49 ± 0.44 5.15 ± 1.42 4.94 ± 1.12 4.85 ± 0.67 Heart1.05 ± 0.24 1.08 ± 0.32 0.88 ± 0.33 0.45 ± 0.08 Lung 1.68 ± 0.09 1.53 ±0.28 1.00 ± 0.18 1.05 ± 0.51 Liver 1.79 ± 0.09 1.27 ± 0.19 1.08 ± 0.321.28 ± 0.33 Spleen 0.84 ± 0.27  0.6 ± 0.09 0.56 ± 0.11 0.5 ± 0.1 Stomach0.39 ± 0.17 0.27 ± 0.18 0.12 ± 0.04 0.15 ± 0.03 Large Intestine 0.29 ±0.09 0.41 ± 0.11 0.22 ± 0.08 0.16 ± 0.03 Blood 2.15 ± 0.5  1.78 ± 0.281.81 ± 0.54 0.92 ± 0.31 Small Intestine 0.54 ± 0.15 0.33 ± 0.1   0.3 ±0.08 0.22 ± 0.07 Kidney 1.98 ± 0.26 1.83 ± 0.57 2.04 ± 0.36 1.47 ± 0.43Muscle 0.22 ± 0.11 0.24 ± 0.08 0.15 ± 0.02 0.11 ± 0.04 Bone 0.23 ± 0.050.32 ± 0.12  0.3 ± 0.07 0.18 ± 0.05

Table 16 shows tumor-to-tissue activity concentration ratios derivedfrom the ⁶⁴Cu-Tz-SarAr pretargeting biodistribution experiment with a 48h accumulation interval as shown in Table 15.

TABLE 16 1 h 4 h 12 h 24 h Tumor/Blood 2.09 ± 0.52  2.9 ± 0.92 2.72 ±1.03 5.27 ± 1.92 Tumor/Heart 4.27 ± 1.05 4.75 ± 1.92 5.62 ± 2.47 10.9 ±2.55 Tumor/Lung 2.67 ± 0.3  3.37 ± 1.11 4.94 ± 1.45 4.61 ± 2.32Tumor/Liver 2.51 ± 0.27 4.06 ± 1.27 4.58 ± 1.69  3.8 ± 1.11 Tumor/Spleen5.34 ± 1.79 8.55 ± 2.69 8.75 ± 2.61  9.8 ± 2.47 Tumor/Stomach 11.57 ±5.25  18.92 ± 13.29 41.88 ± 18.35 32.81 ± 8.37  Tumor/Large Intestine15.48 ± 5.25  12.68 ± 4.87  22.66 ± 10.19 29.58 ± 6.11  Tumor/SmallIntestine 8.35 ± 2.48 15.71 ± 6.48  16.47 ± 5.62  21.72 ± 7.59 Tumor/Kidney 2.27 ± 0.37 2.81 ± 1.16 2.42 ± 0.7  3.31 ± 1.07Tumor/Muscle 20.34 ± 10.25 21.84 ± 9.2  33.88 ± 9.31  44.18 ± 17.61Tumor/Bone 19.48 ± 4.94  15.86 ± 7.12  16.24 ± 5.16  26.55 ± 7.58 

Table 17 shows the biodistribution of ⁶⁴Cu-Tz-SarAr pretargetingexperiment with a 120 h accumulation interval. Female athymic nude micebearing subcutaneous SW1222 (right shoulder) xenografts (100-150 mm³,18-21 days post-inoculation) were administered 100 μg (0.66 nmol)huA33-TCO (in 200 μL 0.9% sterile saline) via intravenous tail veininjection. After an accumulation interval of 120 h, the same mice werethen administered ⁶⁴Cu-Tz-SarAr (300-350 μCi in 200 μL 0.9% sterilesaline) via intravenous tail vein injection (t=0). The specific activityof the radiotracer was adjusted using cold ^(nat)Cu-Tz-SarAr such thatthe molar ratio of Tz_(injected):huA33_(injected)=1:1. Animals (n=4 pergroup) were euthanized by CO₂(g) asphyxiation at 1, 4, 12, and 24 hafter injection. After asphyxiation, tissues were removed, rinsed inwater, dried in air for 5 min, weighed, and counted in a gamma countercalibrated for ⁶⁴Cu. Counts were converted into activity using acalibration curve generated from known standards. Count data werebackground- and decay-corrected to the time of injection, and thepercent injected dose per gram (% ID/g) for each tissue sample wascalculated by normalization to the total activity injected. Contentswithin the stomach, small intestine, and large intestine were notremoved for the measurements.

TABLE 17 1 h 4 h 12 h 24 h Blood 1.39 ± 0.19 1.05 ± 0.29 0.44 ± 0.140.75 ± 0.23 Tumor 3.61 ± 0.45 3.55 ± 0.74 3.29 ± 0.40 4.34 ± 0.98 Heart0.52 ± 0.06 0.43 ± 0.11 0.34 ± 0.20 0.40 ± 0.16 Lung 1.41 ± 0.21 0.91 ±0.22 0.59 ± 0.27 0.85 ± 0.29 Liver 1.65 ± 0.07 1.16 ± 0.39 0.96 ± 0.091.06 ± 0.16 Spleen 0.60 ± 0.13 0.50 ± 0.05 0.34 ± 0.14 0.69 ± 0.17Stomach 0.32 ± 0.13 0.25 ± 0.12 0.09 ± 0.03 0.15 ± 0.08 Large Intestine0.29 ± 0.16 0.20 ± 0.13 0.09 ± 0.03 0.16 ± 0.04 Small Intestine 0.34 ±0.03 0.22 ± 0.03 0.15 ± 0.07 0.17 ± 0.06 Kidney 1.80 ± 0.12 2.10 ± 0.202.11 ± 0.13 1.90 ± 0.17 Muscle 0.20 ± 0.03 0.11 ± 0.05 0.08 ± 0.04 0.14± 0.05 Bone 0.21 ± 0.03 0.16 ± 0.04 0.11 ± 0.04 0.25 ± 0.08

Table 18 shows tumor-to-tissue activity concentration ratios derivedfrom the ⁶⁴Cu-Tz-SarAr pretargeting biodistribution experiment with a120 h accumulation interval as shown in Table 17.

TABLE 18 1 h 4 h 12 h 24 h Tumor/Blood 2.61 ± 0.48 3.37 ± 1.16 7.46 ±2.54 5.74 ± 2.17 Tumor/Heart 7.00 ± 1.22 8.24 ± 2.71 9.56 ± 5.64 10.76 ±4.94  Tumor/Lung 2.55 ± 0.49 3.89 ± 1.23 5.55 ± 2.62 5.09 ± 2.06Tumor/Liver 2.19 ± 0.29 3.06 ± 1.20 3.43 ± 0.52 4.09 ± 1.13 Tumor/Spleen6.02 ± 1.46 7.03 ± 1.64 9.63 ± 4.03 6.26 ± 2.09 Tumor/Stomach 11.37 ±4.88  14.04 ± 7.07  38.07 ± 12.42 29.21 ± 17.97 Tumor/Large Intestine12.39 ± 7.14  17.82 ± 12.22 35.43 ± 12.17 26.48 ± 8.29  Tumor/SmallIntestine 10.53 ± 1.58  16.22 ± 3.91  21.47 ± 9.60  25.34 ± 10.4 Tumor/Blood 2.61 ± 0.48 3.37 ± 1.16 7.46 ± 2.54 5.74 ± 2.17 Tumor/Kidney2.01 ± 0.29 1.69 ± 0.39 1.56 ± 0.21 2.29 ± 0.56 Tumor/Muscle 18.41 ±3.47  32.58 ± 16.17 38.74 ± 20.11 30.39 ± 11.94 Tumor/Bone 17.27 ± 3.03 21.53 ± 7.14  29.07 ± 11.14 17.16 ± 6.90 

Table 19 shows a comparison of salient tumor-to-tissue activityconcentration ratios at 1 h post-injection created using the differentpretargeted PET imaging strategies discussed (see Tables 11, 14, 16, and18).

TABLE 19 Radioligand ⁶⁴Cu-Tz- NOTA ⁶⁴Cu-Tz-SarAr Accumulation Interval24 h 24 h 48 h 120 h Tumor/Blood 1.17 ± 0.22 1.34 ± 0.3  2.09 ± 0.522.61 ± 0.48 Tumor/Liver 1.86 ± 0.24 3.52 ± 0.49 2.51 ± 0.27 2.19 ± 0.29Tumor/Large Intestine 0.31 ± 0.08 26.88 ± 12.11 15.48 ± 5.25  12.39 ±7.14  Tumor/Kidney 3.14 ± 0.41 1.83 ± 0.27 2.27 ± 0.37 2.01 ± 0.29Tumor/Muscle 18.42 ± 3.71  14.92 ± 2.85  20.34 ± 10.25 18.41 ± 3.47 

Table 20 shows a comparison of salient tumor-to-tissue activityconcentration ratios at 4 h post-injection created using the differentpretargeted PET imaging strategies discussed (see Tables 11, 14, 16, and18).

TABLE 20 Radioligand ⁶⁴Cu-Tz- NOTA ⁶⁴Cu-Tz-SarAr Accumulation Interval24 h 24 h 48 h 120 h Tumor/Blood 1.57 ± 0.55 1.39 ± 0.26  2.9 ± 0.923.37 ± 1.16 Tumor/Liver 3.23 ± 0.9  3.83 ± 0.83 4.06 ± 1.27 3.06 ± 1.2 Tumor/Large Intestine 0.43 ± 0.2  11.65 ± 2.99  12.68 ± 4.87  17.82 ±12.22 Tumor/Kidney 4.32 ± 1.56 2.00 ± 0.53 2.81 ± 1.16 1.69 ± 0.39Tumor/Muscle 29.38 ± 8.5  14.84 ± 3.69  21.84 ± 9.2  32.58 ± 16.17

Table 21 shows a comparison of salient tumor-to-tissue activityconcentration ratios at 12 h post-injection created using the differentpretargeted PET imaging strategies discussed (see Tables 11, 14, 16, and18).

TABLE 21 Radioligand ⁶⁴Cu-Tz- NOTA ⁶⁴Cu-Tz-SarAr Accumulation Interval24 h 24 h 48 h 120 h Tumor/Blood 1.82 ± 0.48 3.07 ± 0.79 2.72 ± 1.037.46 ± 2.54 Tumor/Liver 4.53 ± 1.45 5.06 ± 2.23 4.58 ± 1.69 3.43 ± 0.52Tumor/Large Intestine 1.44 ± 0.72 33.67 ± 8.82  22.66 ± 10.19 35.43 ±12.17 Tumor/Kidney  4.6 ± 1.71 3.61 ± 1.05 2.42 ± 0.7  1.56 ± 0.21Tumor/Muscle 26.63 ± 6.59  45.12 ± 8.55  33.88 ± 9.31  38.74 ± 20.11

Table 22 shows a comparison of salient tumor-to-tissue activityconcentration ratios at 24 h post-injection created using the differentpretargeted PET imaging strategies discussed (see Tables 11, 14, 16, and18).

TABLE 22 Radioligand ⁶⁴Cu-Tz- NOTA ⁶⁴Cu-Tz-SarAr Accumulation Interval24 h 24 h 48 h 120 h Tumor/Blood  1.9 ± 0.63 2.83 ± 0.81 5.27 ± 1.925.74 ± 2.17 Tumor/Liver 3.69 ± 1.01 4.87 ± 1.48  3.8 ± 1.11 4.09 ± 1.13Tumor/Large Intestine 2.36 ± 1.37 28.86 ± 10.28 29.58 ± 6.11  26.48 ±8.29  Tumor/Kidney 5.61 ± 1.98 3.68 ± 1.1  3.31 ± 1.07 2.29 ± 0.56Tumor/Muscle 26.98 ± 7.41  37.37 ± 16.06 44.18 ± 17.61 30.39 ± 11.94

Two trends were observed. First, the activity concentrations in thetumor were decreased using longer intervals. For example, the activityconcentrations decreased from 5.6±0.9% ID/g at 4 h post-injection usinga 24 h interval to 5.2±1.4% ID/g and 3.6±0.7% ID/g at the sametime-point using 48 and 120 h intervals, respectively (FIG. 18C). It isthought that this is because the TCO moiety is not infinitely stable totrans-cis isomerization (and thus inactivation) in vivo. However, inboth cases the amount of uptake in the tumor at 1 h post-injection wasnearly identical to that at 24 h, suggesting that longer accumulationintervals effectively eliminated click ligations in the blood. Second,the activity concentrations in the blood and most other tissues werereduced (FIG. 18D). The activity concentration remaining in the blood at4 h post-injection with a 24 h accumulation interval was 4.0±0.37% ID/g.Using 48 and 120 h intervals, the corresponding values decreased to1.78±0.28% ID/g and 1.05±0.29% ID/g, respectively. Taken together, thesetwo trends resulted in higher tumor-to-blood activity ratios for thelonger accumulation intervals, for example, 2.83±0.81, 5.27±1.92, and5.74±2.17 at 24 h post-injection for 24, 48, and 120 h intervals,respectively (FIG. 18E). Tumor-to-background activity ratios remainedlowed in non-target tissue because activity concentrations were offsetby the decreases in the activity concentrations in the tumor (FIG. 18F).

Autoradiography and Immunohistochemistry

Immediately following the pretargeted PET imaging studies, ex vivoautoradiographical and immunohistochemical analyses were performed onthe SW1222 xenografts in order to learn more about the microscopicdistribution of the huA33-TCO and ⁶⁴Cu-Tz-SarAr (FIG. 25). As expected,hematoxylin and eosin staining of the excised tumors revealed that bothhuA33-TCO and ⁶⁴Cu-Tz-SarAr were almost exclusively associated withtumor cells rather than regions of stromal tissue. Moreover, microscopicco-localization of the autoradiographical signal of ⁶⁴Cu-Tz-SarAr andthe fluorescence staining for huA33-TCO was demonstrated. Theseobservations further supported the selective, in vivo formation of thecompleted ⁶⁴Cu-SarAr huA33 radioimmunoconjugate.

Dosimetry

The radiation dosimetry of ⁶⁴Cu-Tz-SarAr was performed to ensure theselective delivery of radioisotopes to malignant tissues at radiationdoses below the doses of traditional radioimmunoconjugates. To this end,dosimetry calculations were performed using the biodistribution datacollected and the OLINDA computer program to determine the mean organabsorbed doses (rad/mCi) and effective dose (rem/mCi) for each strategy(Table 23). The total effective dose of the ⁶⁴Cu-Tz-SarAr pretargetingstrategy with a 24 h interval period was 0.041 rem/mCi, a slightreduction compared to the 0.046 rem/mCi effective dose created by⁶⁴Cu-Tz-NOTA. This difference was due to the significant reduction inthe mean organ absorbed dose to the large intestine. Moreover, the totaleffective dose of the ⁶⁴Cu-Tz-SarAr pretargeting strategy was inverselyproportional to the duration of the accumulation interval. For example,the effective dose decreased from 0.041 rem/mCi with a 24 h interval to0.038 rem/mCi with a 48 h interval to 0.034 with a 120 h interval.

Table 23 shows dosimetry calculations for various huA33-based PETimaging strategies.

TABLE 23 Pretargeting ⁶⁴Cu-Tz- ⁸⁹Zr- ⁶⁴Cu- NOTA* ⁶⁴Cu-Tz-SarAr TargetDFO NOTA 24 h 24 h 48 h 120 h Organ^(†) huA33* huA33* interval intervalinterval interval Adrenals 1.64 0.0726 0.0251 0.0374 0.0348 0.0322 Brain0.764 0.0555 0.0236 0.0361 0.0339 0.0316 Breasts 0.621 0.0509 0.02090.0323 0.0303 0.0281 Gallbladder 1.44 0.0741 0.0272 0.0391 0.0368 0.0341Wall Lower Lg. 1.34 0.193 0.166 0.0482 0.0456 0.0400 Int. Wall Small1.11 0.0832 0.033 0.0418 0.0390 0.0362 Intestine Stomach 0.949 0.08830.0267 0.0552 0.0422 0.0394 Wall Upper Lg. 1.20 0.145 0.114 0.04090.0382 0.0355 Int. Wall Heart Wall 1.55 0.108 0.0294 0.0427 0.03790.0330 Kidneys 2.53 0.186 0.0315 0.0661 0.0539 0.0566 Liver 2.84 0.1940.0311 0.0303 0.0334 0.0301 Lungs 2.26 0.179 0.0289 0.0325 0.0326 0.0216Muscle 1.27 0.0546 0.0138 0.0193 0.0163 0.0139 Ovaries 1.09 0.06810.0299 0.0401 0.0374 0.0346 Pancreas 1.37 0.0708 0.0258 0.0395 0.03640.0338 Red Marrow 3.12 0.308 0.0530 0.0301 0.0280 0.0258 Osteogenic 6.090.439 0.0852 0.0893 0.0805 0.0720 Cells Skin 0.677 0.0464 0.0194 0.02970.0277 0.0258 Spleen 2.52 0.120 0.0180 0.0370 0.0205 0.0225 Adrenals1.64 0.0726 0.0251 0.0374 0.0348 0.0322 Testes 0.683 0.0522 0.02250.0345 0.0322 0.0299 Thymus 0.988 0.0584 0.0227 0.0349 0.0325 0.0299Thyroid 0.947 0.0563 0.0228 0.0351 0.0328 0.0303 Bladder Wall 0.8260.0609 0.0262 0.0391 0.0365 0.0338 Uterus 0.941 0.0652 0.0277 0.04100.0383 0.0355 Total Body 1.39 0.0855 0.0272 0.0379 0.0348 0.0316Effective 1.54 0.133 0.046 0.0414 0.0377 0.0341 Dose ^(†)Mean organabsorbed doses and effective dose are expressed in rad/mCi and rem/mCi,respectively. *Data originally reported in Zeglis, B. M. et al. Journalof Nuclear Medicine. 54, 1389-1396 (2013). ©2013 by the Society ofNuclear Medicine and Molecular Imaging, Inc.

Next, the dosimetry of the ⁶⁴Cu-Tz-SarAr pretargeting strategy anddirectly labeled antibodies was compared. Using a 120 h accumulationinterval, the ⁶⁴Cu-Tz-SarAr pretargeting approach yielded an effectivedose of 0.034 rem/mCi, a nearly four-fold reduction compared to the0.133 rem/mCi effective dose delivered by huA33 labeled directly with⁶⁴Cu. Moreover, the effective doses of ⁶⁴Cu-Tz-SarAr to huA33 directlylabeled with ⁸⁹Zr was compared. In this case, the effective dose of⁸⁹Zr-DFO huA33 was 1.54 rem/mCi, or almost 50 times greater than the0.034 rem/mCi dose associated with ⁶⁴Cu-Tz-SarAr pretargeting with a 120h accumulation interval. In some tissues, this dose rate reduction wasmore pronounced. For example, the mean absorbed doses to osteogeniccells and red marrow with ⁸⁹Zr-DFO huA33 was 6.09 rad/mCi and 3.12rad/mCi, respectively, approximately 80 and 120 times higher than thedose delivered to the same tissues by the ⁶⁴Cu-Tz-SarAr pretargetingapproach.

Experimental Examples of ⁶⁴Cu Tetrazine Radioligands Methods andMaterials

Unless otherwise noted, all chemicals were acquired from Sigma-Aldrich(St. Louis, Mo.) and were used as received without further purification.All water employed was ultra-pure (greater than 18.2 M⁻¹ cm⁻¹ at 25°C.), all DMSO was of molecular biology grade (greater than 99.9%), andall other solvents were of the highest grade commercially available.Acetonitrile (CH3CN) and dimethylformamide (DMF) were purchased fromAcros Organics (Waltham, Mass.) as extra dry over molecular sieves.Amine-reactive trans-cyclooctene [(E)-cyclooct-4-enyl2,5-dioxo-1-pyrrolidinyl carbonate; TCO-NHS)] and amine-reactivetetrazine (2,6-dioxo-1-pyrrolidinyl5-[4-(1,2,4,5-tetrazin-3-yl)benzylamino]-5-oxopentanoate; Tz-NHS) werepurchased from Sigma-Aldrich (St. Louis, Mo.). p-NCS-Bn-NOTA,p-NH2-Bn-NOTA, and DiAmSar chelators were purchased from Macrocyclics,Inc. (Dallas, Tex.). Tz-NOTA and 64Cu-Tz-NOTA were synthesized aspreviously reported. 44 Humanized A33 (huA33) antibody was generouslyprovided by the Ludwig Institute for Cancer Research (New York, N.Y.)and stored at −80° C. prior to use. ⁶⁴Cu was purchased from WashingtonUniversity, St. Louis, where it was produced on a medical cyclotron(Model CS-15, Cyclotron Corp.) via the ⁶⁴Ni(p,n)⁶⁴Cu transformation andpurified as previously described to yield [⁶⁴Cu]CuCl₂ with an effectivespecific activity of 200-400 mCi/μg. 45 Human colorectal cancer cellline SW1222 was obtained from the Ludwig Institute for CancerImmunotherapy and grown by serial passage. Amine-reactive AlexaFluor®680 (AF680-NHS) was purchased from ThermoFisher Scientific (Waltham,Mass.).

Synthesis of tert-butyl(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,7-dioxo-11,14,17,20,23,26,29heptaoxa-2,8-diazahentriacontan-31-yl)carbamate (Tz-PEG7-NHBoc)

Tz-NHS (10 mg; 0.025 mmol; 398.4 g/mol) was dissolved in 400 μL DMSO andadded to 15 mg O-(2-aminoethyl)-O′-[2-(bocamino)ethyl]hexaethyleneglycol (0.032 mmol; 1.3 equiv.; 468.6 g/mol). 10 μL triethylamine (7.3mg; 0.072 mmol; 101.2 g/mol) was then added to this solution, and thesolution was placed on an agitating thermomixer at 300 rpm for 30minutes at room temperature. After 30 minutes, the reaction was purifiedvia preparative C18 HPLC using a gradient of 5:95 MeCN:H2O (both with0.1% TFA) to 95:5 MeCN:H2O over 30 min (tR=18.2 min). Lyophilization ofthe HPLC eluent yielded the purified product as a 16 mg of a bright pinkpowder (MW=751.9 g/mol; 0.021 mmol; 85% yield). 1H NMR (500 MHz, DMSO),δ, ppm: 10.52 (s, 1H), 8.50 (m, 3H), 7.82 (t, 1H), 7.46 (d, 2H), 6.69(t, 1H), 4.33 (d, 2H), 3.42 (m, 22H), 3.33 (t, 2H), 3.31 (t, 2H), 3.12(q, 2H), 2.99 (q, 2H), 2.12 (t, 2H), 2.03 (t, 2H), 2.12 (t, 2H), 1.70(q, 2H), 1.29 (s, 9H). ESI-MS(+): m/z (%)=753.1 [M+H]+ HRMS (ESI): m/zcalcd. for C35H57N7O11Na: 774.4005. found: 774.4014. UV-Vis: ε₅₂₅=530M⁻¹ cm⁻¹.

Synthesis ofN¹-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-N⁵-(23-amino-3,6,9,12,15,18,21heptaoxatricosyl)glutaramide (Tz-PEG₇-NH₂)

Tz-PEG₇-NHBoc (10 mg; 0.014 mmol; 717.5 g/mol) was dissolved in 400 μLof 1:1 CH₂Cl₂:TFA and placed on an agitating thermomixer at 300 rpm for30 minutes at room temperature. After 30 minutes, the solvent wasremoved via rotary evaporation, the residue was taken back up in H₂O,and the reaction was purified via preparative C₁₈ HPLC using a gradientof 5:95 MeCN:H₂O (both with 0.1% TFA) to 95:5 MeCN:H₂O over 30 min(t_(R)=12.5 min). Lyophilization of the HPLC eluent yielded the purifiedproduct as 9 mg of a bright pink powder (MW=651.7; 0.013 mmol; 95%yield). ¹H NMR (500 MHz, DMSO), δ, ppm: 10.58 (s, 1H), 8.46 (m, 2H),7.87 (t, 1H), 7.75 (d, 2H), 7.52 (d, 1H), 4.40 (d, 2H), 3.60-3.50 (m,26H), 3.40 (t, 2H), 3.32 (bs, 2H), 3.20 (q, 2H), 2.99 (bs, 2H), 2.19 (t,2H), 2.12 (t, 2H), 1.79 (q, 2H). ESI-MS(+): m/z (%)=652.9 [M+H]⁺ HRMS(ESI): m/z calcd. for C₃₀H₅₀N₇O₉: 652.3670. found: 652.3676. UV-Vis:ε₅₂₅=535 M⁻¹ cm⁻¹.

Synthesis of2,2′,2″-(2-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,7-dioxo-11,14,17,20,23,26,29heptaoxa-2,8-diazahentriacontan-31-yl)thioureido)benzyl)-1,4,7-triazonane-1,4,7-triyl)triaceticacid (Tz-PEG₇-NOTA)

Tz-PEG₇-NH₂ (5 mg; 0.008 mmol; 651.8 g/mol) was dissolved in 400 μL DMSOand added to 10 mg p-NCS-Bn-NOTA (0.022 mmol; 2.75 equiv.; 450.5 g/mol).10 μL triethylamine (7.3 mg; 0.072 mmol; 101.2 g/mol) was then added tothis solution, and the solution was placed on an agitating thermomixerat 300 rpm for 30 minutes at room temperature. After 30 minutes, thereaction was purified via preparative C₁₈ HPLC using a gradient of 5:95MeCN:H₂O (both with 0.1% TFA) to 95:5 MeCN:H₂O over 30 min (t_(R)=15.5min). Lyophilization of the HPLC eluent yielded the purified product as6 mg of a bright pink powder (MW=1102.2; 0.005 mmol; 68% yield). ¹H NMR(500 MHz, DMSO), δ, ppm: 10.51 (s, 1H), 9.50 (bs, 1H), 8.40 (m, 3H),7.79 (m, 1H), 7.62 (m, 1H), 7.47 (d, 2H), 7.35 (d, 2H), 7.03 (d, 2H),4.43 (d, 2H), 4.00-3.20 (m, 50H), 3.12 (q, 2H), 2.96 (bs, 2H), 2.11 (t,2H), 2.03 (t, 2H), 1.70 (q, 2H). ESI-MS(−): m/z (%)=1100.6 [M−H]⁻; 549.9[M−2H]²⁻ HRMS (ESI): m/z calcd. for C₅₀H₇₆N₁₁O₁₅S: 1102.5243. found:1102.5253. UV-Vis: ε₅₂₅=540 M⁻¹ cm⁻¹.

Synthesis of N¹-(4-(((pivaloyloxy)amino)methyl)benzyl)-3,6,10,13,16,19exaazabicyclo[6.6.6]icosane-1,8-diamine (SarAr-Bn-NHBoc)

N-Boc-4-(bromomethyl)-benzylamine (0.037 g; 0.12 mmol; 1.3 equiv.; 300.2g/mol) was added to a stirred solution of DiAmSar (0.030 g; 0.094 mmol;1.0 equiv.; 314.5 g/mol) in anhydrous dimethylformamide (4.0 mL) at roomtemperature. Sodium carbonate (0.034 g; 0.32 mmol; 3.5 equiv.; 105.9g/mol) was added, and the reaction solution was stirred at 70° C. for 16h. The reaction was diluted with water (6.0 mL), and purification byHPLC (3.0 mL/min, 5% to 80% CH₃CN in 15 min) afforded SarAr-Bn-NHBoc(MW=533.8; 0.035 g; 70%) as a colorless solid: t_(R)=9.4 min. ¹H NMR(500 MHz, DMSO), δ, ppm: 7.38-7.50 (m, 4H), 4.18 (m, 2H), 2.31-3.98 (m,42H), 1.35 (s, 9H). ESI-MS(+): m/z=534.5 [M+H]⁺. HRMS (ESI): m/z calcd.for C₂₇H₅₂N₉O₂: 534.4244. found: 534.4250.

Synthesis of N¹-(4-(aminomethyl)benzyl)-3,6,10,13,16,19hexaazabicyclo[6.6.6]icosane-1,8-diamine (SarAr-Bn-NH₂)

Trifluoroacetic acid (2.0 mL) was added slowly to a stirred solution ofSarAr-Bn-NHBoc (0.031 g; 0.058 mmol; 1.0 equiv.; 533.4 g/mol) in dryacetonitrile (2.0 mL), and the reaction mixture was stirred at roomtemperature for 90 min. Evaporation of the solvents under reducedpressure and purification by HPLC (6.0 mL/min, 5% to 60% CH₃CN in 20min) afforded SarAr-Bn-NH₂ (MW=434.7; 0.026 g, 99%) as a colorlesssolid: t_(R)=6.8 min. ¹H NMR (500 MHz, DMSO), δ, ppm: 7.55 (d, 2H), 7.51(d, 2H), 4.23 (s, 2H), 2.56-4.05 (m, 27H). ESI-MS(+): m/z=434.4 [M+H]⁺.HRMS (ESI): m/z calcd. for C₂₂H₄₄N₉: 434.3720. found: 434.3715.

Synthesis ofN¹-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-N⁵-(4-(((8-amino-3,6,10,13,16,19hexaazabicyclo[6.6.6]icosan-1-yl)amino)methyl)benzyl)glutaramide(Tz-SarAr)

A solution of Tz-NHS (5.0 mg; 0.013 mmol; 1.0 equiv.; 398.4 g/mol) inanhydrous dimethylformamide (400 μL) was added to a stirred solution ofSarAr-Bn-NH₂ (5.4 mg; 0.013 mmol; 1.0 equiv.; 434.7 g/mol) in anhydrousdimethylformamide (200 μL) at room temperature, and the reactionsolution was stirred in the dark for 2 h at room temperature. Afterdilution with water (1.8 mL), purification by HPLC (1.0 mL/min, 5% to80% CH₃CN in 15 min) afforded Tz-SarAr (MW=716.9; 3.9 mg; 42%) as a pinksolid: t_(R)=9.5 min. ¹H NMR (600 MHz, D₂O), δ, ppm: 10.25 (s, 1H), 8.31(d, 2H), 7.46 (d, 2H), 7.22-7.27 (m, 4H), 4.39 (m, 2H), 4.24 (m, 3H),2.46-3.95 (m, 24H), 2.41-2.44 (m, 4H), 1.83 (m, 2H). ESI-MS(+):m/z=717.6 [M+H]⁺. HRMS (ESI): m/z calcd. for C₃₆H₅₇N₁₄O₂: 717.4789.found: 717.4788.

Preparation of ⁶⁴ Cu-Tz-PEG₇-NOTA

A solution of Tz-PEG₇-NOTA (5-25 μg; 4.5-22.6 nmol) in NH₄OAc buffer(0.2 M, pH 5.5, 200 μL) was first prepared. Then, the desired amount of⁶⁴CuCl₂ in 0.1 M HCl (1500-7500 μCi) was added to the reaction mixture,and the solution was placed on an agitating thermomixer at 300 rpm for30 minutes at room temperature. After this incubation, the⁶⁴Cu-Tz-PEG₇-NOTA was purified via reverse phase C₁₈ HPLC (t_(R)=9.7min) to yield the completed radioligand in greater than 99%radionuclidic purity, 78±6% decay-corrected isolated yield, and aspecific activity of 278±32 μCi/μg (323±37 mCi/μmol).

Preparation of ⁶⁴ Cu-Tz-SarAr

A solution of Tz-SarAr (5-25 μg; 6.9-34.9 nmol) in NH₄OAc buffer (0.2 M,pH 5.5, 200 μt) was first prepared. Then, the desired amount of ⁶⁴CuCl₂in 0.1 M HCl (1500-7500 μCi) was added to the reaction mixture, and thesolution was placed on an agitating thermomixer at 300 rpm for 30minutes at room temperature. After this incubation, the⁶⁴Cu-Tz-PEG₇-NOTA was purified via reverse phase C₁₈ HPLC (t_(R)=8.7min) to yield the completed radioligand in greater than 99%radionuclidic purity, 79±7% decay-corrected isolated yield, and aspecific activity of 398±46 μCi/μg (310±36 mCi/μmol) (n=6).

Pretargeted PET Imaging Experiments

All pretargeted PET imaging experiments were performed on an InveonPET/CT scanner (Siemens Healthcare Global). Female athymic nude mice(n=5 per radioligand) bearing subcutaneous SW1222 (right shoulder)xenografts (100-150 mm³, 9-12 days post-inoculation) were administered100 μg (0.66 nmol) huA33-TCO (in 200 μL 0.9% sterile saline) viaintravenous tail vein injection. After an accumulation interval of 24,48, or 120 h, the same mice were then administered either⁶⁴Cu-Tz-PEG₇-NOTA or ⁶⁴Cu-Tz-SarAr (400-450 μCi in 200 μL 0.9% sterilesaline), also via intravenous tail vein injection (t=0). For both⁶⁴Cu-Tz-PEG₇-NOTA and ⁶⁴Cu-Tz-SarAr, the specific activity of theradiotracer was adjusted using cold ^(nat)Cu-Tz-PEG₇-NOTA or^(nat)Cu-Tz-SarAr such that the molar ratio ofTz_(injected):huA33_(injected)≈1:1. Approximately 5 minutes prior to thePET images, mice were anesthetized by inhalation of 2% isoflurane(Baxter Healthcare, Deerfield, Ill.)/oxygen gas mixture and placed onthe scanner bed; anesthesia was maintained using 1% isoflurane/gasmixture. Static scans were recorded at various time points afterinjection with a minimum of 30 million coincident events (10-30 mintotal scan time). An energy window of 350-700 keV and a coincidencetiming window of 6 ns were used. Data were sorted into 2-dimensionalhistograms by Fourier re-binning, and the images were reconstructedusing a two-dimensional ordered subset expectation maximization (2DOSEM)algorithm (16 subsets, 4 iterations) into a 128×128×159 (0.78×0.78×0.80mm) matrix. The image data was normalized to correct for non-uniformityof response of the PET, dead-time count losses, positron branchingratio, and physical decay to the time of injection, but no attenuation,scatter, or partial-volume averaging correction was applied. Activityconcentrations (percentage of dose per gram of tissue [% ID/g]) andmaximum intensity projections were determined by conversion of thecounting rates from the reconstructed images. All of the resulting PETimages were analyzed using ASIPro VM™ software. Whole-body CT scans wereacquired with a voltage of 80 kV and 500 μA. 120 rotational steps for atotal of 220° were acquired with a total scan time of 120 s and 145 msper frame exposure. Combined PET/CT images were processed using InveonResearch Workplace software.

Pretargeted Biodistribution Experiments

Female athymic nude mice bearing subcutaneous SW1222 (right shoulder)xenografts (100-150 mm³, 9-12 days post-inoculation) were administered100 μg (0.66 nmol) huA33-TCO (in 200 μL 0.9% sterile saline) viaintravenous tail vein injection. After an accumulation interval periodof 24, 48, or 120 h, the same mice were then administered ⁶⁴Cu-Tz-SarAr(300-350 μCi in 200 μL 0.9% sterile saline), also via intravenous tailvein injection (t=0). As in the PET imaging experiments, the specificactivity of the radiotracer was adjusted using cold ^(nat)Cu-Tz-SarArsuch that the molar ratio of Tz_(injected):huA33_(injected)=1:1. Animals(n=4 per group) were euthanized by CO₂(g) asphyxiation at 1, 4, and 24 hafter injection. After asphyxiation, tissues were removed, rinsed inwater, dried in air for 5 min, weighed, and counted in a gamma countercalibrated for ⁶⁴Cu. Counts were converted into activity using acalibration curve generated from known standards. Count data werebackground- and decay-corrected to the time of injection, and thepercent injected dose per gram (% ID/g) for each tissue sample wascalculated by normalization to the total activity injected.

Instrumentation

All instruments were calibrated and maintained in accordance withstandard quality-control procedures. UV-Vis measurements were taken on aThermo Scientific NanoDrop 2000 Spectrophotometer. NMR spectroscopy wasperformed on a Bruker 500 MHz NMR with TopSpin 2.1 software for spectrumanalysis. Electrospray ionization mass spectrometry (ESI-MS) spectrawere recorded with a Waters Acquity UPLC (Milford, Calif.) withelectrospray ionization SQ detector. High-resolution mass spectrometry(HRMS) spectra were recorded with a Waters LCT Premier system (ESI).Activity measurements were made using a Capintec CRC-15R Dose Calibrator(Capintec, Ramsey, N.J.). For accurate quantification of activities,experimental samples were counted for 1 min on a calibrated Perkin Elmer(Waltham, Mass.) Automatic Wizard Gamma Counter. Labeling of antibodieswith ⁶⁴Cu-labeled tetrazine radioligands was monitored using silica-gelimpregnated glass-fiber instant thin-layer chromatography paper (PallCorp., East Hills, N.Y.) and analyzed on a Bioscan AR-2000 radio-TLCplate reader using Winscan Radio-TLC software (Bioscan Inc., Washington,D.C.).

HPLC

All HPLC purifications (6.0 mL/min, Buffer A: 0.1% TFA in water, BufferB: 0.1% TFA in CH₃CN) were performed on a Shimadzu UFLC HPLC systemequipped with a DGU-20A degasser, a SPD-M20A UV detector, a LC-6AB pumpsystem, a CBM-20A communication BUS module, and a FRC-10A fractioncollector using a C₁₈ reversed phase XTerra® Preparative MS OBD™ column(10 μm, 19.2 mm×250 mm) or a C₁₈ reversed phase semi-Prep Phenomenex®Jupiter column (5 μm, 10 mm×250 mm). Quality controls of synthesizedcompounds were performed using a C₁₈ reversed phase Atlantis® T3 column(5 μm, 4.6 mm×250 mm). All radio-HPLC analysis and purificationexperiments were performed using a Shimadzu HPLC equipped with a C₁₈reversed phase column (Phenomenex Luna analytical 4.6×250 mm), 2 LC-10ATpumps, a SPD-M10AVP photodiode array detector, a Bioscan Flow Countsradioactivity detector, and a gradient of 5:95 CH₃CN:H₂O (both with 0.1%TFA) to 95:5 CH₃CN:H₂O over 15 min.

Synthesis of Tz-PEG₇-AF680

Tz-PEG₇-NH₂ (1 mg; 0.0015 mmol; 651.8 g/mol) was dissolved in 400 μLDMSO and added to 2 mg AF680-NHS (0.0017 mmol; 1.1 equiv.; ˜1150 g/mol).10 μL triethylamine (7.3 mg; 0.072 mmol; 101.2 g/mol) was then added tothis solution, and the solution was placed on an agitating thermomixerat 300 rpm for 30 minutes at room temperature. After 30 minutes, thereaction was purified via preparative C₁₈ HPLC using a gradient of 5:95CH₃CN:H₂O (both with 0.1% TFA) to 95:5 CH₃CN:H₂O over 30 min (t_(R)=11.2min). Lyophilization of the HPLC eluent yielded the purified product asa 2 mg of a bright orange powder (MW ˜1750; ˜0.0011 mmol; ˜75% yield).

Preparation of huA33-TCO

huA33 (2 mg, 13.3 nmol) was dissolved in 500 μL of phosphate bufferedsaline (PBS, pH 7.4), and the pH of the solution was adjusted to 8.8-9.0with NaHCO₃ (0.1 M). To this solution was added an appropriate volume ofTCO-NHS in DMF (10 mg/mL) to yield a TCO-NHS:huA33 reactionstoichiometry of 10:1. The resulting solution was incubated with gentleshaking for 30 min at room temperature. After 30 min, the modifiedantibody was purified using size-exclusion chromatography (Sephadex G-25M, PD-10 column, GE Healthcare; dead volume=2.5 mL, eluted with 500 mLfractions of PBS, pH 7.4) and concentrated with centrifugal filtrationunits with a 50,000 molecular weight cut off (Amicon™ Ultra 4, MilliporeCorp., Billerica, Mass.) and PBS (pH 7.4).

Determination of the TCO Occupancy of huA33-TCO

A solution of huA33-TCO (100 μg; 0.66 nmol) in 900 μL PBS (pH 7.4) wasfirst prepared (0.74 μM). To this solution, 100 μL of a 1 mM solution ofTz-PEG₇-AF680 in DMSO was added to create a reaction solution of 1000 μLand concentrations of 0.66 μM huA33-TCO and 100 μM Tz-PEG₇-AF680 (a ˜150fold excess of Tz). This solution was placed on an agitating thermomixerat 300 rpm for 180 minutes at room temperature. After incubation, theresulting fluorophore-labeled immunoconjugate was purified usingsize-exclusion chromatography (Sephadex G-25 M, PD-10 column, GEHealthcare; dead volume=2.5 mL, eluted with 500 mL fractions of PBS, pH7.4) and concentrated with centrifugal filtration units with a 50,000molecular weight cut off (Amicon™ Ultra 4, Millipore Corp., Billerica,Mass.) and PBS (pH 7.4). The degree of labeling (DOL) was determined viaUV-Vis. Absorbance measurements were taken at 280 nm and 680 nm forthree separate antibody concentrations. The DOL was calculated using thefollowing formulas:

A _(mAb) =A ₂₈₀ −A _(max)(CF)

DOL=[A _(max)*MW_(mAb)]/[[mAb]*ε_(AF680)]

where the correction factor (CF) for AF680 was given as 0.05 by thesupplier, MW_(huA33)=150,000, ε_(AF680)=184,000, andε_(280, mAb)=225,000. Given the rapid and quantitative nature of theIEDDA reaction, the degree of labeling of AF680 was assumed to be thedegree of labeling of TCO.

Determination of Partition Coefficient

⁶⁴Cu-Tz-PEG₇-NOTA, ⁶⁴Cu-Tz-NOTA, or ⁶⁴Cu-Tz-SarAr (1 μCi) was added to amixture of 3 mL PBS (pH 7.4) and 3 mL 1-octanol. The resulting mixturewas then vortexed thoroughly for 10 minutes and subsequently centrifugedat 1,000 rpm for 10 min. 1 mL of each layer (PBS and 1-octanol) was thencollected, and the amount of radioactivity in each sample was counted ona gamma counter calibrated for ⁶⁴Cu. The partition coefficient (log D)was calculated using the formula:

Log D=log₁₀ [(counts_(octanol))/(counts_(PBS)]

All experiments were performed in triplicate.Reaction of ⁶⁴ Cu-Tz Radioligands with huA33-TCO

In order to check their reactivity with TCO, ⁶⁴Cu-Tz-PEG₇-NOTA and⁶⁴Cu-Tz-SarAr were added to a solution of huA33 in PBS (500 μL, pH 7.4)at a molar ratio of Tz:mAb of 1:1. The resulting solution was placed onan agitating thermomixer at 300 rpm for 30 minutes at room temperature.After this incubation, the progress of the reaction was assayed usingradio-TLC with reverse-phase C₁₈ TLC plates and a mobile phase of 1:1CH₃CN:water (each with 0.1% TFA). Under these conditions, the⁶⁴Cu-labeled antibody will remain at the baseline, while the⁶⁴Cu-Tz-labeled radioligands will travel up the plate. If the purifiedradioimmunoconjugate is desired, the ⁶⁴Cu-Tz huA33 was then purifiedusing size-exclusion chromatography (Sephadex G-25 M, PD-10 column, GEHealthcare; dead volume=2.5 mL, eluted with 500 mL fractions of PBS, pH7.4). Typically, crude radiochemical yields of 90-95% were obtained, andpost-purification radiochemical purities were greater than 99%(corresponding to specific activities of 2.0-2.5 mCi/mg). Allexperiments were performed in triplicate.

PBS and Serum Stability of ⁶⁴Cu-Tz-PEG₇-NOTA and ⁶⁴Cu-Tz-SarAr

⁶⁴Cu-Tz-PEG₇-NOTA and ⁶⁴Cu-Tz-SarAr (1000 μCi) were incubated on anagitating thermomixer (300 rpm) at 37° C. in 500 μL of either PBS (pH7.4) or human serum. At each prescribed time-point, 100 μL of thesolution was removed and placed into a 1.7 mL microcentrifuge tube. Forthe PBS samples, the compound was injected directly onto the HPLC andanalyzed using a gradient of 5:95 CH₃CN:H₂O (both with 0.1% TFA) to 95:5CH₃CN:H₂O over 15 min. For the serum samples, 100 μL cold CH₃CN wasadded to the serum, and the resultant mixture was vortexed andcentrifuged at 10,000 rpm for 10 min. After this, the clear supernatantwas removed, moved to a new 1.7 mL microcentrifuge tube, and centrifugedagain at 10,000 rpm for 10 min. The clear supernatant from this secondspin was then injected into the HPLC and analyzed using a gradient of5:95 CH₃CN:H₂O (both with 0.1% TFA) to 95:5 CH₃CN:H₂O over 15 min. Theresidual protein from the centrifuge spins was checked forradioactivity, and only minimal residual activity remained (less than 1%of the starting Cu for each ⁶⁴Cu-Tz-PEG₇-NOTA and ⁶⁴Cu-Tz-SarAr). Thefraction of intact ⁶⁴Cu-Tz-PEG₇-NOTA or ⁶⁴Cu-Tz-SarAr was determined byintegrating the peak corresponding to the compound (t_(R)=9.7 and 8.7minutes, respectively) and dividing by the integral over the whole HPLCrun. Both the injection loop and column were monitored to detect thepresence of residual activity. All experiments were performed intriplicate.

In Vivo Stability of ⁶⁴Cu-Tz-SarAr

Healthy athymic nude mice were injected with ⁶⁴Cu-Tz-SarAr (300-350 μCi)via intravenous tail vein injection. After 15 min, 1 h, or 4 h, the micewere sacrificed via CO₂ asphyxiation, and their blood (500 μL) wascollected via cardiac puncture in heparinized 1.7 mL microcentrifugetubes. These tubes were then centrifuged at 10,000 rpm for 10 minutes toseparate plasma from red-blood cells. After 10 minutes, the plasmasupernatant was then transferred to a new 1.7 mL microcentrifuge tubeand placed on ice. Subsequently, 500 μL of ice-cold CH₃CN was added tothe plasma to precipitate the proteins, and the tubes were vortexedbriefly and centrifuged again for 10 min at 10,000 rpm. After thiscentrifugation, the supernatant was carefully removed and transferred toanother 1.7 mL microcentrifuge tube, in which it was subjected toanother round of centrifugation at 10,000 rpm for 10 min. After thisfinal centrifugation, the supernatant was again transferred to a clean1.7 mL microcentrifuge tube. This solution was then analyzed viaradio-TLC methods using reverse-phase C₁₈ TLC plates and a mobile phaseof 1:1 CH₃CN:water (each with 0.1% TFA). Using this method, any free⁶⁴Cu²⁺ will remain at the baseline, while ⁶⁴Cu-Tz-SarAr and othermetabolites will travel up the TLC plate. The fraction of intact⁶⁴Cu-Tz-SarAr was calculated by dividing the integral of the parentcompound peak over the integral of the entire radio-TLC chromatogram.All experiments were performed in triplicate.

Cell Culture

Human colorectal cell line SW1222 was obtained from the Ludwig Instituteof Cancer Research and maintained in Iscove's Modified Dulbecco'sMedium, supplemented with 10% heat-inactivated fetal calf serum, 2.0 mMglutamine, 100 units/mL penicillin, and 100 units/mL streptomycin in a37° C. environment containing 5% CO₂. Cell lines were harvested andpassaged weekly using a formulation 0.25% trypsin/0.53 mM EDTA in Hank'sBuffered Salt Solution without calcium and magnesium.

Xenograft Models

All animal experiments we performed under an Institutional Animal Careand Use Committee-approved protocol, and the experiments followedinstitutional guidelines for the proper and humane use of animals inresearch. Six to eight week-old athymic nude female mice were obtainedfrom Charles River Laboratories (Wilmington, Mass.). Animals were housedin ventilated cages, were given food and water ad libitum, and wereallowed to acclimatize for approximately 1 week prior to inoculation.SW1222 tumors were induced on the right shoulder by a subcutaneousinjection of 5.0×10⁶ cells in a 150 μL cell suspension of a 1:1 mixtureof fresh media:BD Matrigel (BD Biosciences, Bedford, Ma). The xenograftsreached ideal size for imaging and biodistribution (˜100-150 mm³) inapproximately 18-21 days.

Immunoreactivity Assays

Immunoreactivity assays employing the huA33-TCO antibodies labeled withthe ⁶⁴Cu-Tz radioligands were performed as previously reported using A33antigen-expressing SW1222 human colorectal cancer cells. All experimentswere performed in triplicate.

PET Imaging with ⁶⁴ Cu-Labeled Tetrazine Radioligands

All PET imaging experiments were performed on an Inveon PET/CT scanner(Siemens Healthcare Global). Healthy female athymic nude mice (n=4 perradioligand) were administered ⁶⁴Cu-Tz-NOTA, ⁶⁴Cu-Tz-PEG₇-NOTA, or⁶⁴Cu-Tz-SarAr (300-350 μCi in 200 μL 0.9% sterile saline) viaintravenous tail vein injection (t=0). Approximately 5 minutes prior tothe PET images, mice were anesthetized by inhalation of 2% isoflurane(Baxter Healthcare, Deerfield, Ill.)/oxygen gas mixture and placed onthe scanner bed; anesthesia was maintained using 1% isoflurane/gasmixture. Static scans were recorded at various time points afterinjection with a minimum of 30 million coincident events (10-30 mintotal scan time). An energy window of 350-700 keV and a coincidencetiming window of 6 ns were used. Data were sorted into 2-dimensionalhistograms by Fourier re-binning, and the images were reconstructedusing a two-dimensional ordered subset expectation maximization (2DOSEM)algorithm (16 subsets, 4 iterations) into a 128×128×159 (0.78×0.78×0.80mm) matrix. The image data was normalized to correct for non-uniformityof response of the PET, dead-time count losses, positron branchingratio, and physical decay to the time of injection but no attenuation,scatter, or partial-volume averaging correction was applied. Activityconcentrations (percentage of dose per gram of tissue [% ID/g]) andmaximum intensity projections were determined by conversion of thecounting rates from the reconstructed images. All of the resulting PETimages were analyzed using ASIPro VM™ software.

Acute Biodistribution with ⁶⁴Cu-Labeled Tetrazine Radioligands

Healthy female athymic nude mice (n=4 per radioligand) were administered⁶⁴Cu-Tz-PEG₇-NOTA or ⁶⁴Cu-Tz-SarAr (25-30 μCi in 200 μL, 0.9% sterilesaline) via intravenous tail vein injection (t=0). Animals (n=4 pergroup) were euthanized by CO₂(g) asphyxiation at 1, 4, 12, and 24 hafter injection. After asphyxiation, tissues were removed, rinsed inwater, dried in air for 5 min, weighed, and counted in a gamma countercalibrated for ⁶⁴Cu. Counts were converted into activity using acalibration curve generated from known standards. Count data werebackground- and decay-corrected to the time of injection, and thepercent injected dose per gram (% ID/g) for each tissue sample wascalculated by normalization to the total activity injected.

Ex Vivo Autoradiography, Immunohistochemistry, and Histology

Following PET imaging, tumors were excised and embedded inoptimal-cutting-temperature mounting medium (OCT, Sakura Finetek) andfrozen on dry ice. Series of 10 μm frozen sections were then cut. Todetermine radiotracer distribution, digital autoradiography wasperformed by placing tissue sections in a film cassette against aphosphor imaging plate (Fujifilm BAS-MS2325; Fuji Photo Film) for anappropriate exposure period at −20° C. Phosphor imaging plates were readat a pixel resolution of 25 μm with a Typhoon 7000 IP plate reader (GEHealthcare). After autoradiographic exposure, the same frozen sectionswere then used for fluorescence staining and microscopy.Immunofluorescence staining and imaging of A33 was performed essentiallyas previously described by Oehler et al. in “¹⁸F-fluromisonidazole PETimaging as a biomarker for the response to5,6-dimethylxanthenone-4-acetic acid in colorectal xenograft tumors.Journal of Nuclear Medicine 2011, 52 (3), 437-44. Frozen sections werefixed in 4% paraformaldehyde, and subsequently incubated with huA33primary antibody (5 μg/ml) overnight at 4° C., followed by secondarydetection using goat anti human Alexa-568 for 1 h at room temperature(20 μg/ml, Molecular Probes). Whole mount fluorescence images wereacquired at ×40 magnification using a BX60 fluorescence microscope(Olympus America, Inc.) equipped with a motorized stage (PriorScientific Instruments Ltd.) and CC12 camera (Olympus). Whole-tumormontage images were obtained by acquiring multiple fields at ×40magnification, followed by alignment using MicroSuite Biologic Suite(version 2.7; Olympus). Fluorescence and autoradiographic images wereregistered using Adobe Photoshop (CS6).

Dosimetry

Mouse biodistribution data were expressed as normal-organ mean standarduptake values (SUVs) versus time post-administration. In first order,that SUVs were independent of body mass and thus the same among species,the mean SUV in mouse organ i, SUV_(Organ i|Mouse), was converted to thefraction of the injected dose in each human organ I,FID_(Organ I|Human), using the following formula:

${FID}_{{{Organ}\mspace{14mu} i}|{Human}} = {{SUV}_{{{ran}\mspace{14mu} i}|{Mouse}} \cdot \frac{{Mass}\mspace{14mu} {of}\mspace{14mu} {Human}\mspace{14mu} {Organ}\mspace{14mu} i}{{Mass}\mspace{14mu} {of}\mspace{14mu} {Human}\mspace{14mu} {Total}\mspace{14mu} {Body}}}$

and the organ and total-body masses of the 70-kg Standard Man anatomicmodel. These data (corrected for radioactive decay to the time ofinjection) were then fit to a mono-exponential or bi-exponentialtime-activity function, depending on the organ. The cumulated activity,or residence time, in human organ i, μC_(i), in μC_(i) h/μC_(i), wasthen calculated by analytically integrating the time-activity functionin organ i, replacing the biological clearance constant, (λ_(b)λ_(j).for each component j of the fitted exponential function with thecorresponding effective clearance constant,(λ_(e)λ_(j)=(λ_(b)κ_(j)+λ_(p), where λ_(p) is the physical decayconstant of the radionuclide. The resulting organ residence times wereentered into the OLINDA computer program to yield the mean organabsorbed doses and effective dose in rad/mCi and rem/mCi, respectively.

Statistical Analysis

Data were analyzed by the unpaired, two-tailed Student's t-test.Differences at the 95% confidence level (P less than 0.05) wereconsidered to be statistically significant.

¹⁸F-Based of Tetrazine Radioligands for Pretargeted Imaging

Described herein is the development of strategies for pretargeted PETimaging of pancreatic cancer featuring a tetrazine-bearing radioligandwith an improved pharmacokinetic profile compared to prior systems. ATCO-bearing immuno-conjugate of the anti-CA19.9 antibody 5B1 and anAl[18^(F)]-NOTA-labeled tetrazine radioligand were harnessed for thevisualization of CA19.9-expressing BxPC3 pancreatic cancer xenografts.Biodistribution and ¹⁸F-PET imaging data clearly demonstrate that thismethodology effectively delineates tumor mass with activityconcentrations up to 6.4% ID/g at 4 h after injection of theradioligand.

The development of a novel Tz/TCO-based pretargeting strategy using anAl[¹⁸F]-NOTA-labeled tetrazine radioligand is described herein. Asdescribed below, the 5B1 antibody, a fully-human IgG that targets apromising biomarker for pancreatic ductal adenocarcinoma: carbohydrateantigen 19.9 (CA19.9) was selected. In order to functionalize theantibody with a reactive bioorthogonal moiety, purified 5B1 wasincubated with an activated succinimidyl ester of TCO (TCO-NHS, 35 eq.)at room temperature for 1 h. The immunoconjugate was subsequentlypurified by gel-filtration chromatography. The precursor to theradioligand, Tz-PEG₁₁-NOTA (FIG. 27, 1), was synthesized from threecommercially available building blocks: (i) 2,5-dioxo-1-pyrrolidinyl5-[4-(1,2,4,5-tetrazin-3-yl)benzylamino]-5-oxopentanoate (Tz-NHS), (ii)O-(2-aminoethyl)-O′-[2-(boc-amino)ethyl]decaethylene glycol(NH₂-PEG₁₁-NHBoc), and (iii)S-2-(4-isothiocyanatobenzyl)-1,4,7-triaza-cyclononane-1,4,7-triaceticacid (p-SCN-Bn-NOTA). After the peptide coupling between Tz-NHS andNH₂-PEG₁₁-NHBoc and the subsequent deprotection of the terminaltert-butyloxycarbonyl protecting group, the resulting Tz-PEG₁₁-NH₂moiety was reacted with the bifunctional p-SCN-Bn-NOTA chelator.Ultimately, the precursor was prepared in very high purity (greater than98%) and with an overall yield of ˜15% (n=3).

The ¹⁸F-labeled radioligand Tz-PEG₁₁-Al[¹⁸F]-NOTA ([¹⁸F]2 (FIG. 27, 2)was obtained in 54-65% radiochemical yield [decay-corrected (d.c.) tothe start of synthesis] in high purity (greater than 96%) and had aspecific activity between 21.4-26.7 GBq/μmol. The use of metal-freesolvents, the pH of the Al[¹⁸F]-NOTA complexation reaction (pH=4), andthe ratio of reaction solvents (at least 3:1 MeCN/H₂O) all proved to becrucial factors in obtaining high radiochemical yields. Aqueous[¹⁸F]fluoride (non-carrier added) was obtained from the cyclotron andloaded onto a preconditioned anion-exchange (QMA) cartridge. Beforeelution of the [¹⁸F]fluoride using 0.4 M KHCO₃-solution (0.2 mL), thecartridge was washed with metal-free water (10-15 mL) to elute metalions present in the original target water. Metal-free glacial acid(˜15-20 μL) was used to adjust the pH to ˜3.5-4, followed by theaddition of 2 mM AlCl₃-solution (25 μL). The resulting mixture wasincubated at room temperature for the formation of the Al-¹⁸F complex.Precursor 1 (40 nmol in 700 μL MeCN) was then added to the solution ofAl[¹⁸F], and the resulting mixture was stirred at 90° C. for 15 min.Subsequently, the ¹⁸F-labeled product was purified using a SepPakC18-cartridge (Waters, Milford, Mass.) and eluted with a small volume ofethanol (0.3-0.4 mL). The in vitro stability of [¹⁸F]2 was assayed byincubation in phosphate buffered saline (PBS, pH 7.4) or human serum at37° C., followed by analysis via radio-HPLC. In PBS, negligibledecomposition could be observed after 4 h (92±2.3% intact), and 79±4.4%(n=4) of the radioligand remained intact in human serum at the same timepoint. Given the fast reaction kinetics of the IEDDA ligation as well asthe relatively short half-life of ¹⁸F, the observed degradation rate isnot considered a detriment to the system, as shown for other Tz/TCOapproaches.

The bioorthogonal click reaction between [¹⁸F]2 and the TCO moiety onthe antibody was demonstrated by incubation of equimolar amounts (1.33nmol) of the purified radioligand with 5B1-TCO at room temperature.Analysis of the reaction via radio-TLC (mobile phase: 90% MeCN in H₂O)revealed a greater than 94% yield for the reaction, with the ¹⁸F-labeledclick reaction product situated at the origin, while the freeradioligand can be detected at the solvent front.

In vivo biodistribution data for Tz-PEG₁₁-Al[¹⁸F]-NOTA were firstobtained in healthy mice by injecting [¹⁸F]2 alone (1.8-2.0 MBq) via thetail vein. The data showed accumulation and retention of the radiotracerin the large intestines and feces with 0.32±0.87 percent injected doseper gram (% ID/g) at 1 h after injection to 1.73±0.45 (% ID/g) at 4 h.The uptake and retention of [¹⁸F]2 could also be observed in the kidneys(2.12±0.23% ID/g at 1 h to 1.17±0.12% ID/g at 4 h), indicating dualrenal and fecal elimination pathways for the radioligand. The amount ofactivity in the blood decreased over time, from 1.94±0.23% ID/g at 1 hto 0.78±0.08% ID/g at 4 h after injection, while the uptake in all otherhealthy tissues remained less than 1% ID/g. Notably, the activityconcentrations in the bone were particularly low (not exceeding 0.2%ID/g), illustrating in vivo stability of the Al[¹⁸F]-NOTA complex. Inaccompanying experiments, the blood half-life of the radioligand wascalculated to be 71.2 min.

In subsequent pretargeted biodistribution experiments, nude, athymicmice bearing subcutaneous CA19.9-expressing BxPC3 xenografts wereinjected with 5B1-TCO (1.33 nmol) 72 h prior to the administration of[¹⁸F]2 (1.33 nmol, 1.8-2.0 MBq). The data revealed increasing tumoraluptake over the course of the study (3.0±0.32% ID/g at 30 min,3.52±0.67% ID/g at 1 h, 4.81±1.23% ID/g at 2 h to 5.6±0.85% ID/g at 4h), with the amount of radioactivity in the blood decreasing in kind,from 6.13±0.86% ID/g at 30 min to 1.75±0.22% ID/g at 4 h. In accordanceto the biodistribution data obtained from healthy mice, the uptake inother tissue remained generally low (less than or equal to 2% ID/g),with the highest uptake and retention in the clearance organs: theintestines and kidneys (FIG. 28).

The clearance of radioactivity from the blood pool was generally in linewith the calculated blood half-life of the radiotracer, and the steadyuptake of radioactivity at the tumor suggested that the radioligand isprimarily clicking with 5B1-TCO at the tumor site rather than clickingin the blood pool followed by accumulation at the tumor.

Small animal PET imaging experiments were conducted in a similarfashion, with the only difference in the amount of radioactivityinjected (18-20 MBq, 1.33 nmol of [¹⁸F]2, equimolar to 5B1-TCO). The PETimages as shown in FIG. 29 confirm the data obtained in thebiodistribution study: the signal in the tumor increases with time,while the activity concentrations in the blood and intestinesconcomitantly decrease. This results in the clear delineation of thetumor from background tissue, with the tumor-to-background activityratios improving over the course of the experiment. The tumoral uptakeof [¹⁸F]2 is immediately evident 1 h after injection; however, thesignal grows to 6.4% ID/g at 4 h after the administration of theradioligand. While the tumor-to-background activity concentration ratiosimprove over time, radioactivity had not cleared the intestines at 4 hpost-injection. Therefore, second generation tetrazine-bearingradioligands can be developed in an effort to determine whetherstructural alterations can increase the fraction of the radioligand thatis excreted via the renal system, and thus create highertumor-to-background ratios at earlier time points. Finally, using thebiodistribution data, a dosimetric analysis of the pretargeting strategywas performed that confirms that pretargeted PET imaging withTz-PEG₁₁-Al¹⁸F-NOTA and 5B1-TCO confers a significant dosimetricadvantage over the use of antibodies directly labeled with long-livedradioisotopes (e.g., in the case ⁸⁹Zr-DFO-5B1). The effective dose ofthe presented ¹⁸F-based pretargeting system (0.03 rem/mCi) is more than60 times lower than directly labeled ⁸⁹Zr-DFO-5B1 (2.02 rem/mCi).

Thus, the ¹⁸F-based pretargeted PET imaging system described hereinshows highly promising biodistribution results and produced tumoractivity concentrations of up to 6.4% ID/g at 4 h post-injection.Small-animal PET imaging experiments revealed that this methodologyclearly delineates CA19.9-expressing tissues, with especially enticingtumor-to-background activity ratios 2 h and 4 h after injection of theradiotracer.

All starting materials except the NOTA-Bn-p-NSC that was purchased fromMacrocyclics were purchased from Sigma-Aldrich (synthetic-grade) andwere used without further purification. All solvents used for HPLCanalysis and purification within this project were purchased from FisherScientific (HPLC grade). Metal-free DMSO (greater than or equal to99.99995%) and MeCN (greater than or equal to 99.999%) were purchasedfrom Sigma-Aldrich. Water (greater than 18.2 MΩ cm-1 at 25° C.) wasobtained from an Alpha-Q Ultrapure water system from Millipore (Bedford,Mass.).

Proton (¹H) NMR spectra were measured on a BrukerAvance Ultra Shield(500 MHz) spectrometer at ambient temperature. Data were recorded asfollows: chemical shift in ppm from internal reference tetramethylsilaneon the scale, multiplicity (s=singlet; d=doublet; t=triplet;m=multiplet), coupling constant (Hz), integration, and assignment.Carbon (¹³C) NMR spectra were measured on a BrukerAvance Ultra Shield(125 MHz) spectrometer at ambient temperature. Chemical shifts wererecorded in ppm from the solvent resonance employed as the internalstandard (deuterochloroform at 77.00 ppm).

Non-carrier-added (n.c.a.) ¹⁸F-fluoride was obtained via the ¹⁸O(p,n)¹⁸Fnuclear reaction of 11-MeV protons in an EBCO TR-19/9 cyclotron usingenriched ¹⁸O-water. QMA light ion-exchange cartridges and C-18 lightSep-Pak® cartridges were obtained from Waters (Milford, Mass.). C18cartridges were equilibrated using absolute ethanol (10 mL) followed bydeionized water (5 mL). QMA cartridges used a Chromafix 30-PS-HCO3-resinfor ion-exchange and were equilibrated using KHCO3-solution (0.4 M, 5mL) followed by deionized water (10 mL). High performance liquidchromatography (HPLC) purification and analysis was performed on aShimadzu UFLC HPLC system equipped with a DGU-20A degasser, a SPD-M20AUV detector, a RF-20Axs fluorescence detector, a LC-20AB pump system,and a CBM-20A communication BUS module. A LabLogic Scan-RAMradio-TLC/HPLC-detector was used for purifications while a PosiRAM Model4 was used for analysis. HPLC solvents (Buffer A: 0.1% TFA in water,Buffer B: 0.1% TFA in MeCN) were filtered before use. HPLC analysis ofradioactive and non-radioactive compounds was performed on a reversedphase Atlantis T3 column (C18, 5 μm, 4.6 mm×250 mm). Preparative HPLCpurification was carried out on a reversed phase Waters XTerra Prep C18OBD (C18, 10 μm, 19 mm×250 mm). For radioactive thin-layerchromatography (TLC) analysis throughout this work, Merck precoated TLCplates (C18, reversed-phase) were used. Radio-TLC was performed using aCanberra 190 5 Experimental Part UNISPEC iScan (Meriden, Conn., USA)instrument. Radioactivity was determined using a calibrated ion chamber(Capintec CRC-15R).

Electrospray ionization mass spectrometry (ESI-MS) spectra were recordedwith a Shimadzu LC-2020 with electrospray ionization SQ detector.High-resolution mass spectrometry (ESI-HRMS) was carried out on aMicromass LCT Premier XE using a reversed phase Waters XBridge column(C18, 5 μm, 4.6 mm×50 mm).

Synthesestert-butyl-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,7-dioxo-11,14,17,20,23,26,29,32,35,38,41-undecaoxa-2,8-diazatritetracontan-43-yl)carbamate(Tz-PEG₁₁-NHBoc)

2,5-Dioxo-1-pyrrolidinyl5-[4-(1,2,4,5-tetrazin-3-yl)benzylamino]-5-oxopentanoate (Tz-NHS, 10 mg,0.025 mmol) was dissolved in anhydrous dimethylsulfoxide (DMSO, 0.5 mL)before O-(2-Aminoethyl)-O′-[2-(Boc-amino)ethyl]decaethylene glycol (24.2mg, 0.0375 mmol) and TEA (0.0057 mL, 0.0375 mmol) were added, Thereaction mixture was stirred at room temperature for 45 min Aftercompletion of the reaction (monitored by HPLC, 5% MeCN/H to 95% MeCNover 20 min, R_(t)=14.2 min, 1 mL/min) the product was purified usingpreparative HPLC (5% MeCN/H to 95% MeCN over 20 min R_(t)=14.5 min, 8mL/min) with purity greater than 95%. The product was furnished as apink solid (18.2 mg, 96%). ¹H NMR (500 MHz, DMSO-d₆) δ 10.65 (s, 1H),8.77 (t, J=5.4 Hz, 1H), 8.65-8.57 (m, 2H), 8.16-8.12 (m, 2H), 6.77-6.73(m, 1H), 3.62-3.42 (m, 46H), 3.38 (t, J=6.1 Hz, 3H), 3.07 (q, J=5.8 Hz,2H), 1.38 (s, 9H); MS (ESI) m/z 929.3 [M+H]⁺, HRMS (ESI) calcd. forC₄₃H₇₃N₇NaO₁₅ [=M+Na]⁺ m/z 950.5062. found 950.5034.

N¹-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-N⁵-(35-amino-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-pentatriacontyl)glutaramide(Tz-PEG₁₁-NH₂)

Tz-PEG₁₁-NHBoc (16.3 mg, 0.0216 mmol) was dissolved in dichloromethane(DCM, 0.5 mL) before TFA (0.1 mL) was added drop wise. The resultingsolution was stirred at room temperature for 30 min. The solvent wasremoved under reduced pressure before the deprotected product waspurified via preparative HPLC (5% MeCN/H to 95% MeCN over 20 min,R_(t)=11.6 min, 8 mL/min) with purity greater than 97%. The product wasfurnished as a pink solid (14.4 mg, 92%), ¹H NMR (500 MHz, DMSO-d₆) δ10.59 (s, 1H), 8.74 (t, J=5.2 Hz, 1H), 8.61-8.55 (m, 2H), 8.12-8.09 (m,2H), 6.79-6.74 (m, 1H), 3.68-3.36 (m, 46H), 3.34 (t, J=6.1 Hz, 3H), 3.10(q, J=5.8 Hz, 2H); MS (ESI) m/z 829.2 [M+H]⁺, HRMS (ESI) calcd forC₃₈H₆₆N₇O₁₃ [=M+H]⁺ m/z 828.4719. found 828.4742.

2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,7-dioxo-11,14,17,20,23,26,29,32,35,38,-41-undecaoxa-2,8-diazatritetracontan-43-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid (Tz-PEG₁₁-NOTA)

Tz-PEG₁₁-NH₂ (13.5 mg, 0.0216 mmol) was dissolved in DMSO (0.5 mL)before NOTA-Bn-NCS (20.2 mg, 0.036 mmol) and TEA (0.0057 mL, 0.0375mmol) were added. The reaction mixture was stirred at room temperaturefor 45 min. After completion of the reaction (monitored by HPLC, 5%MeCN/H to 95% MeCN over 20 min, R_(t)=13.2 min, 1 ml/min) the productwas purified using preparative HPLC (5% MeCN/H₂O to 95% MeCN over 30min, R_(t)=13.6 min., 8 mL/min) with purity greater than 97%. Theproduct was furnished as a pink solid (20.2 mg, 73%). ¹H NMR (500 MHz,DMSO-d₆) δ 10.59 (s, 1H), 8.47 (d, J=7.3 Hz, 3H), 7.87 (t, J=5.2 Hz,3H), 7.55 (d, J=7.6 Hz, 3H), 7.43 (d, J=8.1 Hz, 3H), 7.20 (d, J=7.7 Hz,2H), 4.41 (d, J=5.8 Hz, 3H), 4.00 (d, J=17.5 Hz, 2H), 3.82 (d, J=17.9Hz, 4H), 3.51 (s, 53H), 2.20 (t, J=7.4 Hz, 3H), 2.12 (t, J=7.5 Hz, 5H),1.78 (dt, J=14.4, 7.2 Hz, 4H); MS (ESI) m/z 829.2 [M+H]⁺¹, HRMS (ESI)calcd for C₄₃H₇₃N₇O₁₅ [=M−H]⁻ m/z 1276.6135. found 1276.6179.

Tz-PEG₁₁-Al[¹⁸F]-NOTA.

The [¹⁸F]fluoride received from the cyclotron was trapped on apreconditioned QMA cartridge. The cartridge was subsequently washed withmetal-free water (10-15 mL) before the [¹⁸F]fluoride (0.9-1.1 GBq) waseluted using 0.4 M KHCO3-solution (0.2 mL) into a V-vial. The pH of thesolution was adjusted to ˜4 using glacial acetic acid (15-20 μL)followed by the addition of 2 mM AlCl₃-solution (25 μL). The resultingsolution was incubated at room temperature for 20 min to form the Al-¹⁸Fcomplex. The precursor Tz-PEG₁₁-NOTA dissolved in MeCN (700 μL) was thenadded to the aqueous solution containing the Al-¹⁸F complex and theresulting mixture stirred at 90° C. for 15 min. After the given periodof time the reaction vial was cooled using dry ice before the reactionmixture was diluted with water (20 mL). The obtained aqueous solutioncontaining the labeled product was flushed through a preconditioned C18cartridge followed by an additional 10 mL of water to remove left over[¹⁸F]fluoride from the cartridge. The product [¹⁸F]2 was subsequentlyeluted with EtOH (0.3-05 mL) and analyzed for purity using radio-HPLC(5% MeCN/H₂O to 95% MeCN over 20 min, Rt=11.5 min, 1 ml/min). Prior toeach animal experiment the EtOH was removed under reduced pressure andthe tracer was reconstituted in 0.9% saline for injections.

In Vitro Stability Testing

Table 24 shows results obtained from the in vitro stability study of theradioligand [¹⁸F]2. The values are given as percent intact tracer afterincubation.

TABLE 24 Conditions Time PBS (pH = 7.4) [%] Human Serum [%] 30 min 98.5± 1.0 95.5 ± 1.5 1 h 96.5 ± 2.5 91.5 ± 2   2 h   93 ± 1.0   85 ± 4.5 4 h91.5 ± 1.5 74.5 ± 5.5

The radioligand was incubated with agitation (600 rpm) at 37° C. in 500μL of either PBS or human serum. At the appropriate time points, 100 μLof the solution was transferred into a 1.7 mL centrifuge tube. In caseof PBS, the aliquot was directly injected into the HPLC. For the serumsamples, 100 μL of MeCN was added to the previously transferred 100 μLserum solution and the resulting solution was vortexed and centrifuged(13,000 rpm) for 5 min. The clear supernatant was removed, moved to anew centrifuge tube followed by additional centrifugation at 13,000 rpmfor 5 min. The resulting clear supernatant was then used for HPLCanalysis (FIGS. 30A and 30B). The residual protein was checked forradioactivity, and only minimal residual radioactivity could be detected(e.g., less than 2%). The fraction of intact radioligand was calculatedby dividing the peak area corresponding to the tracer by the integral ofthe entire HPLC run. The observed decomposition of the radioligand inhuman serum is likely related to the presence of nucleophilic sulfhydryland amino groups in the serum that could, even at neutral pH, have ameasurable negative effect on the stability of the tetrazineradioligand.

Click Reaction of Tz-PEG₁₁-Al[¹⁸F]-NOTA with TCO-Modified 5B1

The click reaction between the radioligand and the TCO moiety of theTCO-modified 5B1 antibody was demonstrated by incubating an equimolaramount (1.33 nmol) of the purified tracer [¹⁸F]2 with 5B1-TCO in PBS(pH=7.4) at room temperature for 15 min. A small aliquot of the reactionmixture was spotted onto a C18 TLC plate. TLC was performed using 90%MeCN in H₂O as mobile phase and analyzed by radio-TLC. The freeradioligand in a control run could be detected at the solvent front(FIG. 31A) whereas the ¹⁸F-labeled click reaction product (FIG. 31B) wassituated at the origin showing that the click reaction was completedafter 15 min.

Cell Culture

BxPC3 cells were purchased from ATCC (Manassas, Va.) and grown in RPMImodified to contain 4.5 g/L glucose and 1.5 g/L sodium bicarbonate andsupplemented with 10% (v/v) fetal calf serum, 10 mM HEPES, 1 mM sodiumpyruvate, 2 mM L-glutamine, 10 cc/L non-essential amino acids, 100 IUpenicillin and 100 ug streptomycin.

In Vivo Models

All animal experiments within this study were performed in accordancewith protocols approved by the Institutional Animal Care and UseCommittee of MSKCC and followed National Institutes of Health guidelinesfor animal welfare. Female athymic nude CrTac:NCr-Foxn1^(nu) mice at age6-8 weeks were purchased from Charles River Laboratories. Forsubcutaneous injections, mice were anesthetized with 2% isoflurane(Baxter Healthcare) (2 L/min medical air) before BxPC3 cells wereimplanted subcutaneously (5×10⁶ cells in 150 μL 1:1 growthmedia/Matrigel® (BD Biosciences, San Jose, Calif.) in the right shoulderand allowed to grow for approximately 3-4 weeks until the tumors reached5-10 mm in size. For all intravenous injections, mice were gently warmedwith a heat lamp and placed on a restrainer. The tails were sterilizedwith alcohol pads, and injection took place via the lateral tail vein.

Biodistribution in Healthy Nude Mice

The radioligand Tz-PEG₁₁-Al¹⁸F-NOTA was injected into healthy mice viathe tail vein (FIG. 32). The organs were harvested at the appropriatetime points after the animals were euthanized by CO₂ asphyxiation. Thecollected organs were weighed and counted in a WIZARD² automaticγ-counter (PerkinElmer, Boston, Mass.). Generally, radioactivity wastaken and retained in the intestines and kidneys. Blood pool clearanceoccurred slower than anticipated for small molecule radiotracer,however, the clearance pattern was in line with the determined bloodhalf-life of 74.4 minutes.

The identical method was used in case of the pretargeted biodistributionexperiments.

Blood Half-Life Determination of Tz-PEG₁₁-Al[¹⁸ F]-NOTA

The tracer Tz-PEG₁₁-Al[¹⁸F]-NOTA ([¹⁸F]2) was injected into healthy nudemice via the tail vein. Blood was collected through the saphenous veinat the appropriate time points (FIG. 33). The collection tubes wereweighed before and after blood collection and measured for radioactivityin order to calculate the percent injected dose per gram values for eachtime point and sample (n=4 for each time point). The half-life wassubsequently calculated to 74.4 minutes.

Dosimetry

The pretargeted biodistribution data obtained from the utilized mousemodel were first expressed as normal-organ mean standard uptake values(SUVs) versus time post-injection. It was assumed that SUVs are, infirst order, independent of body mass and hence the same among species.The mean SUV in mouse organ in every mouse organ was then used tocalculate the mean SUV of the same organs in a human using the organ andtotal-body masses of the 70 kg Standard Man anatomic model. These datawere then corrected for radioactive decay to the time of injection andsubsequently fitted to a mono-exponential or bi-exponentialtime-activity function, depending on the organ. This information wasused to determine the organ residence times which were then entered intothe OLINDA computer program to yield the mean organ absorbed doses andeffective dose in rad/mCi and rem/mCi, respectively. The data obtainedfrom the herein presented ¹⁸F-based pretargeting system were compared tothe pretargeting system previously described by Viola-Villegas et al.using ⁸⁹Zr-labeled 5B1 for pretargeting CA19.9 (Table 25).

Table 25 shows mean organ absorbed doses and effective dose calculatedfor the herein described pretargeting approach given in rad/mCi andrem/mCi, respectively, compared to the pretargeting system previously byN. T. Viola-Villegas, S. L. Rice, S. Carlin, X. Wu, M. J. Evans, K. K.Sevak, M. Drobjnak, G. Ragupathi, R. Sawada, W. W. Scholz, P. O.Livingston, J. S. Lewis, J. Nucl. Med, 2013, 54, 1876-1882, using⁸⁹Zr-labeled 5B1 for pretargeting CA19.9.

TABLE 25 Target Organ ¹⁸F pretargeting ⁸⁹Zr-DFO-5B1^(#) Adrenals 0.02882.22 Brain 0.0256 1.7 Breasts 0.0214 1.36 Gallbladder Wall 0.0297 2.13LLI Wall 0.0487 2.22 Small Intestine 0.0356 2.1 Stomach Wall 0.0401 2.2ULI Wall 0.0424 1.98 Heart Wall 0.0279 2.15 Kidneys 0.113 2.86 Liver0.0223 2.52 Lungs 0.0181 2.52 Muscle 0.0138 1.59 Ovaries 0.0299 1.98Pancreas 0.0327 2.26 Red Marrow 0.0227 4.01 Osteogenic Cells 0.0366 5.08Skin 0.018 1.17 Spleen 0.0997 3.7 Testes 0.0233 1.51 Thymus 0.0234 1.71Thyroid 0.0235 1.69 Urinary Bladder Wall 0.0286 1.86 Uterus 0.0306 1.99Total Body 0.0235 1.86 Effective dose 0.0302 2.02Exemplary Tetrazine Precursors Available for Radiolabeling with ¹⁸F

Below are listed examples of available tetrazine-based precursormolecules that can be used for radiolabeling with ¹⁸F using theAl[¹⁸F]-NOTA methodology.

Tetrazine Structures Connected to NOTA-Chelators Via PEG-Linkers:

Tetrazine Structures Connected to NOTA/NODA-Chelators Via(Poly)-L-Lysine-Linkers:

1. A composition comprising: a tetrazine moiety (Tz); a radiolabel; achelator; a linker attaching the tetrazine moiety (Tz) to the chelator;and aluminum or aluminum-containing moiety.
 2. The composition of claim1, wherein the linker is polyethylene glycol (PEG) or (poly)-L-lysineand has a length of from 1 to 100 units and 1 to 200 units,respectively.
 3. The composition of claim 1, the composition comprising:


4. A radioimmunoconjugate comprising: (1) a targetingmoiety-transcyclooctene (TCO) conjugate; and (2) a radioligandcomprising a tetrazine moiety (Tz); a radiolabel; a chelator; a linkerattaching the tetrazine moiety (Tz) to the chelator; and aluminum oraluminum-containing moiety.
 5. The composition of claim 1, wherein thetetrazine moiety (Tz), the chelator, and the linker attaching thetetrazine moiety to the chelator comprises a member selected from thegroup consisting of:2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-1-oxo-5,8,11,14,17,20,23-heptaoxa-2-azapentacosan-25-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35-undecaoxa-2-azaheptatriacontan-37-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′-(7-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35-undecaoxa-2-azaheptatriacontan-37-yl)thioureido)benzyl)-1,4,7-triazonane-1,4-diyl)diaceticacid;2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,7-dioxo-11,14,17,20,23,26,29-heptaoxa-2,8-diazahentriacontan-31-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,7-dioxo-11,14,17,20,23,26,29,32,35,38,41-undecaoxa-2,8-diazatritetracontan-43-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(25,28-dioxo-28-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)-3,6,9,12,15,18,21-heptaoxa-24-azaoctacosyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(37,40-dioxo-40-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36-azatetracontyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)-3-oxo-6,9,12,15,18,21,24-heptaoxa-2-azaheptacosan-27-amido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(2-(4-(1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy)-3,6,9,12,15,18,21,24,27,30,33-undecaoxahexatriacontan-36-amido)benzyl)-1,4,7-triazonane-1,4,7-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(5-amino-6-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′-(7-(4-(3-(5-amino-6-((4-6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-triazonane-1,4-diyl)diaceticacid;2,2′,2″-(3-(4-(3-(5-amino-6-((5-amino-6-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid; and2,2′,2″-(3-(4-(3-(5-amino-6-((5-amino-6-((5-amino-6-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)amino)-6-oxohexyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid.
 6. The composition of claim 1, wherein the composition ishydrophilic.
 7. The radioimmunoconjugate of claim 4, the radioligandcomprising:


8. The radioimmunoconjugate of claim 4, wherein the targeting moiety-TCOconjugate has a TCO moiety comprising:


9. The radioimmunoconjugate of claim 4, wherein the linker ispolyethylene glycol (PEG) or (poly)-L-lysine and has a length of from 1to 100 units and 1 to 200 units, respectively.
 10. A method forsynthesizing a radioligand: (1) preparing Tz-PEG₁₁-NOTA(tetrazine-polyethylene glycol-1,4,7-triazonane-1,4,7-triyl-triaceticacid); (2) preparing a Al-¹⁸F complex; and (3) reacting theTz-PEG₁₁-NOTA and the Al-¹⁸F complex to yield the radioligandTz-PEG₁₁-Al[¹⁸F]-NOTA, wherein the radioligand is obtained in 54% to 65%radiochemical yield (decay-corrected to a start of synthesis) with apurity greater than 96% and specific activities between 20 to 30Gbq/μmol.
 11. A method for detecting tumor cells, the method comprising:(1) administering a quantity of targeting moiety-transcyclooctene (TCO)conjugate to a subject, wherein a portion of the targeting moiety-TCOconjugate localizes at the tumor cells and unbound targeting moiety-TCOconjugate is cleared from blood, from renal system, and/or from thesubject after an accumulation interval; (2) administering a radioligandto the subject after the accumulation interval, wherein the radioligandcomprises a tetrazine moiety (Tz); a radiolabel; a chelator; a linkerattaching the tetrazine moiety (Tz) to the chelator; and aluminum oraluminum-containing moiety, wherein the targeting moiety-TCO conjugateand the radioligand bind together to form a radioimmunoconjugate via anin vivo click reaction at the tumor cells within a region of thesubject; and (3) imaging via positron emission tomography (PET) imagingthe radioimmunoconjugate accumulated in the region of the subject withina time period less than 9 hours from the administering of theradioligand.
 12. The method of claim 11, wherein the radioligandcomprises:


13. The method of claim 11, wherein the targeting moiety-TCO conjugatehas a TCO moiety comprising:


14. The method of claim 11, wherein the radioligand has an effectivedose of less than 0.1 rem/mCi over a 4 hour accumulation interval. 15.The method of claim 11, wherein the radioligand has a half-life in bloodthat is less than 100 minutes.
 16. The method of claim 11, wherein theradioimmunoconjugate has an activity concentration in a large intestineof the subject that is less than 2% of an initial dose per gram (ID/g)after 2 hour post injection.
 17. The method of claim 11, wherein theradioimmunoconjugate has an activity concentration in a gastrointestinaltract of the subject that is less than 2% of an initial dose per gram(ID/g) after 2 hour post injection.
 18. The method of claim 11, whereinthe radioimmunoconjugate has an activity concentration in ahepatobiliary system of the subject that is less than 2% of an initialdose per gram (ID/g after 2 hour post injection.
 19. The method of claim11, wherein the linker is polyethylene glycol (PEG) or (poly)-L-lysineand has a length of from 1 to 100 units and 1 to 200 units,respectively.
 20. The method of claim 11, wherein the radioligand ishydrophilic.
 21. The method of claim 11, wherein the targeting moiety isan antibody.
 22. The method of claim 21, wherein the antibody is amember selected from the group consisting of trastuzumab, J591,bevacizumab, B43.13, AR9.6, 3F8, 8H9, huA33, and 5B1.
 23. The method ofclaim 11, wherein the targeting moiety is a nanoparticle, a peptide, orother biomolecule.
 24. The method of claim 11, wherein the tumor cellsare colorectal tumor cells or pancreatic tumor cells.
 25. The method ofclaim 11, wherein the tetrazine moiety (Tz), the chelator, and thelinker attaching the tetrazine moiety to the chelator comprises a memberselected from the group consisting of:2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-1-oxo-5,8,11,14,17,20,23-heptaoxa-2-azapentacosan-25-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35-undecaoxa-2-azaheptatriacontan-37-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′-(7-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35-undecaoxa-2-azaheptatriacontan-37-yl)thioureido)benzyl)-1,4,7-triazonane-1,4-diyl)diaceticacid;2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,7-dioxo-11,14,17,20,23,26,29-heptaoxa-2,8-diazahentriacontan-31-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,7-dioxo-11,14,17,20,23,26,29,32,35,38,41-undecaoxa-2,8-diazatritetracontan-43-yl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(25,28-dioxo-28-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)-3,6,9,12,15,18,21-heptaoxa-24-azaoctacosyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(37,40-dioxo-40-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)-3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36-azatetracontyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(3-(4-(1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)-3-oxo-6,9,12,15,18,21,24-heptaoxa-2-azaheptacosan-27-amido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′,2″-(2-(4-(1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy)-3,6,9,12,15,18,21,24,27,30,33-undecaoxahexatriacontan-36-amido)benzyl)-1,4,7-triazonane-1,4,7-triyl)triaceticacid;2,2′,2″-(3-(4-(3-(5-amino-6-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid;2,2′-(7-(4-(3-(5-amino-6-((4-6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-triazonane-1,4-diyl)diaceticacid;2,2′,2″-(3-(4-(3-(5-amino-6-((5-amino-6-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid; and2,2′,2″-(3-(4-(3-(5-amino-6-((5-amino-6-((5-amino-6-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)amino)-6-oxohexyl)amino)-6-oxohexyl)amino)-6-oxohexyl)thioureido)benzyl)-1,4,7-triazonane-2,5,8-triyl)triaceticacid. 26-45. (canceled)